Steroid derived antibiotics

ABSTRACT

A series of novel steroid derivatives are described. The steroid derivatives are antibacterial agents. The steroid derivatives also act to sensitize bacteria to other antibiotics including erythromycin and novobiocin.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a continuation-in-part of U.S. patent application Ser. No.09/234,008, filed Jan. 19, 1999, which is a continuation-in-part ofPCT/US 98/04489, filed Mar. 6, 1998, each of which is herebyincorporated by reference in its entirety. This application claimspriority from provisional application U.S. 60/225,467, filed Aug. 15,2000, which is hereby incorporated by reference in its entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with support from the National Institutes ofHealth (GM 54619). The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

The invention relates to novel steroid derivatives and processes andintermediates for the preparation of these compounds.

Some compounds that associate strongly with the outer membrane ofGram-negative bacteria are known to disrupt the outer membrane andincrease permeability. The increased permeability can increase thesusceptibility of Gram-negative bacteria to other antibiotics. The beststudied of this type of compound are the polymyxin antibiotics. For anexample of a study involving the binding of polymyxin B to the primaryconstituent of the outer membrane of Gram-negative bacteria (lipid A)see: D. C. Morrison and D. M. Jacobs, Binding of Polymyxin B to TheLipid a Portion of Bacterial Lipopolysaccharides, Immunochemistry 1976,vol. 13, 813-819. For an example of a study involving the binding of apolymyxin derivative to Gram-negative bacteria see: M. Vaara and P.Viljanen, Binding of Polymyxin B Nonapeptide to Gram-negative Bacteria,Antimicrobial Agents and Chemotherapy, 1985, vol. 27, 548-554.

Membranes of Gram-negative bacteria are semipermeable molecular “sieves”which restrict access of antibiotics and host defense molecules to theirtargets within the bacterial cell. Thus, cations and polycations whichinteract with and break down the outer membrane permeability barrier arecapable of increasing the susceptibility of Gram-negative pathogenicbacteria to antibiotics and host defense molecules. Hancock and Wongdemonstrated that a broad range of peptides could overcome thepermeability barrier and coined the name “permeabilizers” to describethem (Hancock and Wong, Antimicrob. Agents Chemother., 26:48, 1984).

SUMMARY OF THE INVENTION

The present invention features compounds of the formula I

-   -   wherein:    -   fused rings A, B, C, and D are independently saturated or fully        or partially unsaturated; and    -   each of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, and R₁₇ is        independently selected from the group consisting of hydrogen,        hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,        (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,        (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)        alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)        alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10)        alkylamino, a substituted or unsubstituted (C1-C10) aminoalkyl,        a substituted or unsubstituted aryl, a substituted or        unsubstituted arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl,        C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a        second steroid, a substituted or unsubstituted (C1-C10)        aminoalkyloxy, a substituted or unsubstituted (C1-C10)        aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted        (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted        (C1-C10) aminoalkylaminocarbonyl, a substituted or unsubstituted        (C1-C10) aminoalkylcarboxamido, H2N—HC(Q5)-C(O)—O—,        H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10)        cyanoalkyloxy, P.G.-HN—C(Q5)-C(O)—O—, (C1-C10) guanidinoalkyl        oxy, (C1-C10) quaternaryammoniumalkylcarboxy, and (C1-C10)        guanidinoalkyl carboxy, where Q5 is a side chain of any amino        acid (including the side chain of glycine, i.e., H), P.G. is an        amino protecting group, and    -   R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is each independently: deleted        when one of fused rings A, B, C, or D is unsaturated so as to        complete the valency of the carbon atom at that site, or    -   selected from the group consisting of hydrogen, hydroxyl, a        substituted or unsubstituted (C1-C10) alkyl, (C1-C10)        hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, a substituted or        unsubstituted (C1-C10) aminoalkyl, a substituted or        unsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6        alkynyl, a linking group attached to a second steroid, a        substituted or unsubstituted (C1-C10) aminoalkyloxy, a        substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a        substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl,        H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—, (C1-C10)        azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,        (C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy,        where Q5 is a side chain of any amino acid, P.G. is an amino        protecting group, and    -   provided that at least two of R₁ through R₁₄ are independently        selected from the group consisting of a substituted or        unsubstituted (C1-C10) aminoalkyloxy, (C1-C10)        alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)        alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10)        alkylamino, a substituted or unsubstituted (C1-C10)        aminoalkylcarboxy, a substituted or unsubstituted        arylamino-(C1-C10) alkyl, a substituted or unsubstituted        (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a substituted or        unsubstituted (C1-C10) aminoalkylaminocarbonyl, (C1-C10)        quaternaryammonium alkylcarboxy, H2N—HC(Q5)-C(O)—O—,        H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10)        cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10)        guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy; or a        pharmaceutically acceptable salt thereof.

The term fused ring used herein can be heterocyclic or carbocyclic,preferably.

The term “saturated” used herein refers to the fused ring of formula Ihaving each atom in the fused ring either hydrogenated or substitutedsuch that the valency of each atom is filled.

The term “unsaturated” used herein refers to the fused ring of formula Iwhere the valency of each atom of the fused ring may not be filled withhydrogen or other substituents. For example, adjacent carbon atoms inthe fused ring can be doubly bound to each other. Unsaturation can alsoinclude deleting at least one of the following pairs and completing thevalency of the ring carbon atoms at these deleted positions with adouble bond; such as R₅ and R₉; R₈ and R₁₀; and R₁₃ and R₁₄.

The term “unsubstituted” used herein refers to a moiety having each atomhydrogenated such that the valency of each atom is filled.

The term “halo” used herein refers to a halogen atom such as fluorine,chlorine, 0.10 bromine, or iodine.

Examples of amino acid side chains include but are not limited to H(glycine), methyl (alanine), —CH₂—(C═O)—NH₂ (asparagine), —CH₂—SH(cysteine), and —CH(OH)CH₃ (threonine).

An alkyl group is a branched or unbranched hydrocarbon that may besubstituted or unsubstituted. Examples of branched alkyl groups includeisopropyl, sec-butyl, isobutyl, tert-butyl, sec-pentyl, isopentyl,tert-pentyl, isohexyl. Substituted alkyl groups may have one, two, threeor more substituents, which may be the same or different, each replacinga hydrogen atom. Substituents are halogen (e.g., F, Cl, Br, and I),hydroxyl, protected hydroxyl, amino, protected amino, carboxy, protectedcarboxy, cyano, methylsulfonylamino, alkoxy, acyloxy, nitro, and lowerhaloalkyl.

The term “substitued” used herein refers to moieties having one, two,three or more substituents, which may be the same or different, eachreplacing a hydrogen atom.

Examples of substituents include but are not limited to halogen (e.g.,F, Cl, Br, and I), hydroxyl, protected hydroxyl, amino, protected amino,carboxy, protected carboxy, cyano, methylsulfonylamino, alkoxy, alkyl,aryl, aralkyl, acyloxy, nitro, and lower haloalkyl.

An aryl group is a C₆₋₂₀ aromatic ring, wherein the ring is made ofcarbon atoms (e.g., C₆₋₁₄, C₆₋₁₀ aryl groups). Examples of haloalkylinclude fluoromethyl, dichloromethyl, trifluoromethyl,1,1-difluoroethyl, and 2,2-dibromoethyl.

An aralkyl group is a group containing 6-20 carbon atoms that has atleast one aryl ring and at least one alkyl or alkylene chain connectedto that ring. An example of an aralkyl group is a benzyl group.

A linking group is any divalent moiety used to link a compound offormula to another steroid, e.g., a second compound of formula I. Anexample of a linking group is (C1-C10) alkyloxy-(C1-C10) alkyl.

Numerous amino-protecting groups are well-known to those in the art. Ingeneral, the species of protecting group is not critical, provided thatit is stable to the conditions of any subsequent reaction(s) on otherpositions of the compound and can be removed at the appropriate pointwithout adversely affecting the remainder of the molecule. In addition,a protecting group may be substituted for another after substantivesynthetic transformations are complete. Clearly, where a compounddiffers from a compound disclosed herein only in that one or moreprotecting groups of the disclosed compound has been substituted with adifferent protecting group, that compound is within the invention.Further examples and conditions are found in T. W. Greene, ProtectiveGroups in Organic Chemistry, (1st ed., 1981, 2nd ed., 1991).

The present invention also includes methods of synthesizing compounds offormula I where at least two of R₁ through R₁₄ are independentlyselected from the group consisting of a substituted or unsubstituted(C1-C10) aminoalkyloxy. The method includes the step of contacting acompound of formula IV,

-   -   where at least two of R₁ through R₁₄ are hydroxyl, and the        remaining moieties on the fused rings A, B, C, and D are defined        for formula I, with an electrophile to produce an alkyl ether        compound of formula IV, wherein at least two of R₁ through R₁₄        are (C1-C10)alkyloxy. The alkyl ether compounds are converted        into an amino precursor compound wherein at least two of R₁        through R₁₄ are independently selected from the group consisting        of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy and the        amino precursor compound is reduced to form a compound of        formula I.

The electrophiles used in the method include but are not limited to2-(2-bromoethyl)-1,3-dioxolane, 2-iodoacetamide, 2-chloroacetamide,N-(2-bromoethyl)phthalimide, N-(3-bromopropyl)phthalimide, andallybromide. The preferred electrophile is allylbromide.

The invention also includes a method of producing a compound of formulaI where at least two of R₁ through R₁₄ are (C1-C10) guanidoalkyloxy. Themethod includes contacting a compound of formula IV, where at least twoof R₁ through R₁₄ are hydroxyl, with an electrophile to produce an alkylether compound of formula IV, where at least two of R₁ through R₁₄ are(C1-C10)alkyloxy. The allyl ether compound is converted into an aminoprecursor compound where at least two of R₁ through R₁₄ areindependently selected from the group consisting of (C1-C10)azidoalkyloxy and (C1-C10) cyanoalkyloxy. The amino precursor compoundis reduced to produce an aminoalkyl ether compound wherein at least twoof R₁ through R₁₄ are (C1-C10) aminoalkyloxy. The aminoalkyl ethercompound is contacted with a guanidino producing electrophile to form acompound of formula I.

The term “guanidino producing electrophile” used herein refers to anelectrophile used to produce a guanidino compound of formula I. Anexample of an guanidino producing electrophile is HSO₃—C(NH)—NH₂.

The invention also includes a method of producing a compound of formulaI where at least two of R₁ through R₁₄ are H2N—HC(Q5)-C(O)—O— and Q5 isthe side chain of any amino acid. The method includes the step ofcontacting a compound of formula IV, where at least two of R₁ throughR₁₄ are hydroxyl, with a protected amino acid to produce a protectedamino acid compound of formula IV where at least two of at least two ofR₁ through R₁₄ are P.G.-HN—HC(Q5)-C(O)—O— and Q5 is the side chain ofany amino acid and P.G. is an amino protecting group. The protectinggroup of the protected amino acid compound is removed to form a compoundof formula I.

The present invention also includes pharmaceutical compositions ofmatter that are useful as antibacterial agents, sensitizers of bacteriato other antibiotics and disrupters of bacterial membranes. Thepharmaceutical compositions can be used to treat humans and animalshaving a bacterial infection. The pharmaceutical compositions caninclude an effective amount of the steroid derivative alone or incombination with other antibacterial agents.

The invention further includes a method of preparing the compound (A):

-   -   by    -   (a) contacting 5β-cholanic acid 3,7,12-trione methyl ester with        hydroxylamine hydrochloride and sodium acetate to form the        trioxime (B):    -   (b) contacting trioxime (B) with NaBH₄ and TiCl₄ to yield        compound (A).

The invention also includes a compound comprising a ring system of atleast 4 fused rings, where each of the rings has from 5-7 atoms. Thering system has two faces, and contains 3 chains attached to the sameface. Each of the chains contains a nitrogen-containing group that isseparated from the ring system by at least one atom; thenitrogen-containing group is an amino group, e.g., a primary aminogroup, or a guanidino group. Preferably, the compound also contains ahydrophobic group, such as a substituted (C3-10) aminoalkyl group, a(C1-10) alkyloxy (C3-10) alkyl group, or a (C1-10) alkylamino(C3-10)alkyl group, attached to the steroid backbone.

For example, the compound may have the formula V, where each of thethree chains containing nitrogen-containing groups is independentlyselected from R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈,defined below.

-   -   where:    -   each of fused rings A, B, C, and D is independently saturated,        or is fully or partially unsaturated, provided that at least two        of A, B, C, and D are saturated, wherein rings A, B, C, and D        form a ring system;    -   each of m, n, p, and q is independently 0 or 1;    -   each of R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈        is independently selected from the group consisting of hydrogen,        hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,        (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,        (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)        alkylamino-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10)        alkylamino, (C1-C10) alkylamino-(C1-C10) alkylamino-(C1-C10)        alkylamino, a substituted or unsubstituted (C1-C10) aminoalkyl,        a substituted or unsubstituted aryl, a substituted or        unsubstituted arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl,        C2-C6 alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a        second steroid, a substituted or unsubstituted (C1-C10)        aminoalkyloxy, a substituted or unsubstituted (C1-C10)        aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted        (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted        (C1-C10) aminoalkylaminocarbonyl, a substituted or unsubstituted        (C1-C10) aminoalkylcarboxamido, H2N—HC(Q5)-C(O)—O—,        H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10)        cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyl        oxy, (C1-C10) quaternaryammoniumalkylcarboxy, and (C1-C10)        guanidinoalkyl carboxy, where Q5 is a side chain of any amino        acid (including the side chain of glycine, i.e., H), P.G. is an        amino protecting group; and    -   each of R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is independently: deleted        when one of fused rings A, B, C, or D is unsaturated so as to        complete the valency of the carbon atom at that site, or        selected from the group consisting of hydrogen, hydroxyl, a        substituted or unsubstituted (C1-C10) alkyl, (C1-C10)        hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, a substituted or        unsubstituted (C1-C10) aminoalkyl, a substituted or        unsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6        alkynyl, a linking group attached to a second steroid, a        substituted or unsubstituted (C1-C10) aminoalkyloxy, a        substituted or unsubstituted (C1-C10) aminoalkylcarboxy, a        substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl,        H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—, (C1-C10)        azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,        (C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy,        where Q5 is a side chain of any amino acid, P.G. is an amino        protecting group, provided that at least three of R₁ through R₄,        R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆, R₁₇, and R₁₈ are disposed on the        same face of the ring system and are independently selected from        the group consisting of a substituted or unsubstituted (C1-C10)        aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)        alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)        alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted        (C1-C10) aminoalkylcarboxy, a substituted or unsubstituted        arylamino-(C1-C10) alkyl, a substituted or unsubstituted        (C1-C10) aminoalkyloxy-(C1-C10) alkyl, a substituted or        unsubstituted (C1-C10) aminoalkylaminocarbonyl, (C1-C10)        quaternaryammonium alkylcarboxy, H2N—HC(Q5)-C(O)—O—,        H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10)        cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10)        guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy; or a        pharmaceutically acceptable salt thereof. Preferably, at least        two, or at least, three, of m, n, p, and q are 1.

Without wishing to be bound to any particular theory, the steroidderivatives described herein act as bacteriostatic and bactericidalagents by binding to the outer cellular membrane of bacteria. Theinteraction between the steroid derivatives and the bacteria membranedisrupts the integrity of the cellular membrane and results in the deathof the bacteria cell. In addition, compounds of the present inventionalso act to sensitize bacteria to other antibiotics; At concentrationsof the steroid derivatives below the corresponding minimumbacteriostatic concentration, the derivatives cause bacteria to becomemore susceptible to other antibiotics by increasing the permeability ofthe outer membrane of the bacteria. Measurements used to quantitate theeffects of the steroid derivatives on bacteria include: measurement ofminimum inhibitory concentrations (MICs), measurement of minimumbactericidal concentrations (MBCs) and the ability of the steroidderivatives to lower the MICs of other antibiotics, e.g., erythromycinand novobiocin.

A person of skill will recognize that the compounds described hereinpreserve certain stereochemical and electronic characteristics found insteroids. The term “same configuration” as used herein refers tosubstituents on the fused steroid having the same stereochemicalorientation. For example substituents R3, R7 and R12 are allβ-substituted or α-substituted. The configuration of the moieties R3,R7, and R12 substituted on C3, C7, and C12 may be important forinteraction with the cellular membrane.

In another aspect, the invention features several methods of using theabove-described compounds. For example, an effective amount of ananti-microbial composition comprising such a compound is administered toa host (including a human host) to treat a microbial infection. Thecompound by itself may provide the anti-microbial effect, in which casethe amount of the compound administered is sufficient to beanti-microbial. Alternatively, an additional anti-microbial substance tobe delivered to the microbial cells (e.g., an antibiotic) is included inthe anti-microbial composition. By facilitating delivery to the targetcells, the compounds can enhance the effectiveness of the additionalantimicrobial substance. In some cases the enhancement may besubstantial. Particularly important target microbes are bacteria (e.g.,Gram-negative bacteria generally or bacteria which have a substantial(>40%) amount of a lipid A or lipid A-like substance in the outermembrane). Other microbes including fungi, viruses, and yeast may alsobe the target organisms.

The compounds can also be administered in other contexts to enhance cellpermeability to introduce any of a large number of different kinds ofsubstances into a cell, particularly the bacterial cells discussedabove. In addition to introducing anti-microbial substances, theinvention may be used to introduce other substances such asmacromolecules (e.g., vector-less DNA).

The invention can also be used to make anti-microbial compositions(e.g., disinfectants, antiseptics, antibiotics etc.) which comprise oneof the above compounds. These compositions are not limited topharmaceuticals, and they may be used topically or in non-therapeuticcontexts to control microbial (particularly bacterial) growth. Forexample, they may be used in applications that kill or control microbeson contact.

In yet another aspect, the invention generally features methods ofidentifying compounds that are effective against a microbe byadministering a candidate compound and a compound according to theinvention the microbe and determining whether the candidate compound hasa static or toxic effect (e.g, an antiseptic, germicidal, disinfectant,or antibiotic effect) on the microbe. Again, bacteria such as thosediscussed above are preferred. This aspect of the invention permitsuseful testing of an extremely broad range of candidate anti-microbialswhich are known to have anti-microbial effect in some contexts, butwhich have not yet been shown to have any effect against certain classesof microbes such as the bacteria discussed above. As described ingreater detail below, this aspect of the invention permits testing of abroad range of antibiotics currently thought to be ineffective againstGram-negative or lipid A-like containing bacteria.

In yet another aspect the invention features compositions which includeone of the above compounds in combination with a substance to beintroduced into a cell such as an antimicrobial substance as describedin greater detail above. The compound and the additional substance maybe mixed with a pharmaceutically acceptable carrier.

Other features or advantages of the present invention will be apparentfrom the following detailed description of several embodiments, and alsofrom the appending claims.

The invention encompasses steroid derivatives that can be made by thesynthetic routes described herein, and methods of treating a subjecthaving a condition mediated by a bacterial infection by administering aneffective amount of a pharmaceutical composition containing a compounddisclosed herein to the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing compounds of the invention.

FIG. 2 is a graph showing the concentrations of compounds of theinvention required to lower the MIC of erythromycin to 1 μg/ml, as wellas MIC and MBC values of each of the compounds.

FIG. 3 is a scheme showing the proposed mechanism of action of cholicacid derivatives.

FIG. 4 is a drawing showing compounds of the invention.

FIG. 5 is a graph showing MIC and MBC values for compounds of theinvention.

FIG. 6 is a graph showing MIC values for compounds of the invention.

FIG. 7 is a drawing showing compound 132.

FIG. 8 is a drawing showing compound 211.

FIG. 9 is a drawing showing compounds 352-354.

FIG. 10 is a drawing showing compound 355.

FIG. 11 is a drawing showing compounds 341-343 and 324-327.

FIG. 12 is a drawing showing compounds 356-358.

DETAILED DESCRIPTION

In general, the present invention provides the compounds of formula Idescribed above. The preparation methods and the MIC and MBC ofcompounds of formula I are described. The cellular membrane permeabilityis also measured and described. Compounds that are useful in accordancewith the invention, as described below, include novel steroidderivatives that exhibit bacteriostatic, bactericidal, and bacterialsensitizer properties. Those skilled in the art will appreciate that theinvention extends to other compounds within the formulae given in theclaims below, having the described characteristics. Thesecharacteristics can be determined for each test compound using theassays detailed below and elsewhere in the literature.

Known compounds that are used in accordance with the invention andprecursors to novel compounds according to the invention can bepurchased, e.g., from Sigma Chemical Co., St. Louis; Aldrich, Milwaukee;Steroids and Research Plus. Other compounds according to the inventioncan be synthesized according to known methods and the methods describedbelow using publicly available precursors.

The compounds of the present invention include but are not limited tocompounds having amine or guanidine groups covalently tethered to asteroid backbone, e.g., cholic acid. Other ring systems can also beused, e.g., 5-member fused rings. Compounds with backbones having acombination of 5- and 6-membered rings are also included in theinvention. The amine or guanidine groups are separated from the backboneby at least one atom, and preferably are separated by at least two,three, or four atoms. The backbone can be used to orient the amine orguanidine groups on one face, or plane, of the steroid. For example, ascheme showing a compound having primary amino groups on one face, orplane, of a backbone is shown below:

The biological activity of the compounds can be determined by standardmethods known to those of skill in the art, such as the “minimalinhibitory concentration (MIC)” assay described in the present examples,whereby the lowest concentration at which no change in optical density(OD) is observed for a given period of time is recorded as MIC. When thecompound alone is tested against a control that lacks the compound, theantimicrobial effect of the compound alone is determined.

Alternatively, “fractional inhibitory concentration (FIC)” is alsouseful for determination of synergy between the compounds of theinvention, or the compounds in combination with known antibiotics. FICscan be performed by checkerboard titrations of compounds in onedimension of a microtiter plate, and of antibiotics in the otherdimension, for example. The FIC is calculated by looking at the impactof one antibiotic on the MIC of the other and vice versa. An FIC of oneindicates that the influence of the compounds is additive and an FIC ofless than one indicates synergy. Preferably, an FIC of less than 0.5 isobtained for synergism. As used herein, FIC can be-determined asfollows: ${FIC} = {\frac{\begin{matrix}{{MIC}\quad\left( {{compound}\quad{in}} \right.} \\\left. {combination} \right)\end{matrix}}{{MIC}\quad\left( {{compound}\quad{alone}} \right)} + \frac{\begin{matrix}{{MIC}\quad\left( {{antibiotic}\quad{in}} \right.} \\\left. {combination} \right)\end{matrix}}{{MIC}\quad\left( {{antibiotic}\quad{alone}} \right)}}$

This procedure permits determination of synergistic effects of thecompound with other compounds. For example, substances that generallymay not be sufficiently effective against certain bacteria at safedosages can be made more effective with the compound of the invention,thus enabling use of the substances against new categories ofinfections. Specifically, many existing antibiotics are effectiveagainst some Gram-positive bacteria, but are not currently indicated totreat Gram-negative bacterial infection. In some cases, the antibioticmay be ineffective by itself against Gram-negative bacteria because itfails to enter the cell. Compounds of the invention may increasepermeability so as to render the antibiotics effective againstGram-negative bacteria.

In addition, fractional inhibitory concentration is also useful fordetermination of synergy between compounds of the invention incombination with other compounds having unknown anti-bacterial activityor in combination with other compounds, e.g., compounds which have beentested and show anti-bacterial activity. For example, compounds of theinvention may increase permeability so as to render compounds lackinganti-bacterial activity effective against bacteria. The FIC can also beused to test for other types of previously unappreciated activity ofsubstances that will be introduced into the cell by means ofpermeability enhancing compounds according to the invention.

While we do not wish to be bound to any single specific theory, and sucha theory is not necessary to practice the invention, one mechanism ofaction is the lipid A interaction of multiple (usually three) moieties,which under physiological conditions are positively charged, e.g.,guanidino or amino moieties. The moieties extend away from the generalplane of the remainder of the molecule, thus mimicking certain aspectsof the structure of polymyxins. In this regard, compounds of theinvention will generally be useful in the way that polymyxins areuseful. For example, polymyxin B (PMB) and polymyxin B nonapeptide(PMBN) are useful for permeabilizing bacterial membranes. Moreover, inregard to systemic administration, those skilled in the art willrecognize appropriate toxicity screens that permit selection ofcompounds that are not toxic at dosages that enhance microbialpermeability.

As noted, the invention also involves topical as well as non-therapeutic(antiseptic, germicidal, or disinfecting) applications in which thecompounds are contacted with surfaces to be treated. The term“contacting” preferably refers to exposing the bacteria to the compoundso that the compound can effectively inhibit, kill, or lyse bacteria,bind endotoxin (LPS), or permeabilize Gram-negative bacterial outermembranes. Contacting may be in vitro, for example by adding thecompound to a bacterial culture to test for susceptibility of thebacteria to the compound. Contacting may be in vivo, for exampleadministering the compound to a subject with a bacterial disorder, suchas septic shock. “Inhibiting” or “inhibiting effective amount” refers tothe amount of compound which is required to cause a bacteriostatic orbactericidal effect. Examples of bacteria which may be inhibited includeE. coli, P. aeruginosa, E. cloacae, S. typhimurium, M. tuberculosis andS. aureus. In addition, the compounds of the invention can be used toinhibit antibiotic-resistant strains of microorganisms.

The method of inhibiting the growth of bacteria may further include theaddition of antibiotics for combination or synergistic therapy. Theappropriate antibiotic administered will typically depend on thesusceptibility of the bacteria such as whether the bacteria isGram-negative or Gram-positive, and will be easily discernable by one ofskill in the art. Examples of particular classes of antibiotics to betested for synergistic therapy with the compounds of the invention (asdescribed above) include aminoglycosides (e.g., tobramycin), penicillins(e.g., piperacillin), cephalosporins (e.g., ceftazidime),fluoroquinolones (e.g., ciprofloxacin), carbepenems (e.g., imipenem),tetracyclines and macrolides (e.g., erythromycin and clarithromycin).The method of inhibiting the growth of bacteria may further include theaddition of antibiotics for combination or synergistic therapy. Theappropriate antibiotic administered will typically depend on thesusceptibility of the bacteria such as whether the bacteria isGram-negative or Gram-positive, and will be easily discernable by one ofskill in the art. Further to the antibiotics listed above, typicalantibiotics include aminoglycosides (amikacin, gentamicin, kanamycin,netilmicin, tobramycin, streptomycin, azithromycin, clarithromycin,erythromycin, erythromycin estolate/ethylsuccinate,gluceptate/lactobionate/stearate, beta-lactams such as penicillins(e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin,cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin,carbenicillin, mezlocillin, azlocillin and piperacillin), orcephalosporins (e.g., cephalothin, cefazolin, cefaclor, cefamandole,cefoxitin, cefuroxime, cefonicid, cefinetazole, cefotetan, cefprozil,loracarbef, cefetamet, cefoperazone, cefotaxime, ceftizoxime,ceftriaxone, ceftazidime, cefepime, cefixime, cefpodoxime, andcefsulodin). Other classes of antibiotics include carbapenems (e.g.,imipenem), monobactams (e.g., aztreonam), quinolones (e.g., fleroxacin,nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin,lomefloxacin and cinoxacin), tetracyclines (e.g., doxycycline,minocycline, tetracycline), and glycopeptides (e.g., vancomycin,teicoplanin), for example. Other antibiotics include chloramphenicol,clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampinand mupirocin, and polymyxins, such as PMB.

Administration

The compounds may be administered to any host, including a human ornon-human animal, in an amount effective to inhibit not only growth of abacterium, but also a virus or fungus. These compounds are useful asantimicrobial agents, antiviral agents, and antifingal agents. Thecompounds may be administered to any host, including a human ornon-human animal, in an amount effective to inhibit not only growth of abacterium, but also a virus or fungus. These compounds are useful asantimicrobial agents, antiviral agents, and antifingal agents.

The compounds of the invention can be administered parenterally byinjection or by gradual infusion over time. The compounds can beadministered topically, intravenously, intraperitoneally,intramuscularly, subcutaneously, intracavity, or transdermally.Preferred methods for delivery of the compound include orally, byencapsulation in microspheres or proteinoids, by aerosol delivery to thelungs, or transdermally by iontophoresis or transdermal electroporation.Other methods of administration will be known to those skilled in theart.

Preparations for parenteral administration of a compound of theinvention include sterile aqueous or non-aqueous solutions, suspensions,and emulsions. Examples of non-aqueous solvents are propylene glycol,polyethylene glycol, vegetable oils such as olive oil, and injectableorganic esters such as ethyl oleate. Aqueous carriers include water,alcoholic/aqueous solutions, emulsions or suspensions, including salineand buffered media. Parenteral vehicles include sodium chloridesolution, Ringer's dextrose, dextrose and sodium chloride, lactatedRinger's, or fixed oils. Intravenous vehicles include fluid and nutrientreplenishers, electrolyte replenishers (such as those based on Ringer'sdextrose), and the like. Preservatives and other additives may also bepresent such as, for example, antimicrobials, anti-oxidants, chelatingagents, and inert gases and the like.

The invention provides a method of treating or ameliorating anendotoxemia or septic shock (sepsis) associated disorder, or one or moreof the symptoms of sepsis comprising administering to a subjectdisplaying symptoms of sepsis or at risk for developing sepsis, atherapeutically effective amount of a compound of the invention. Theterm “ameliorate” refers to a decrease or lessening of the symptoms ofthe disorder being treated. Such symptoms which may be amelioratedinclude those associated with a transient increase in the blood level ofTNF, such as fever, hypotension, neutropenia, leukopenia,thrombocytopenia, disseminated intravascular coagulation, adultrespiratory distress syndrome, shock and multiple organ failure.Patients who require such treatment include those at risk for or thosesuffering from toxemia, such as endotoxemia resulting from aGram-negative bacterial infection, venom poisoning, or hepatic failure,for example. In addition, patients having a Gram-positive bacterial,viral or fungal infection may display symptoms of sepsis and may benefitfrom such a therapeutic method as described herein. Those patients whoare more particularly able to benefit from the method of the inventionare those suffering from infection by E. coli, Haemophilus influenza B,Neisseria meningitidis, staphylococci, or pneumococci. Patients at riskfor sepsis include those suffering from gunshot wounds, renal or hepaticfailure, trauma, burns, immunocompromised (HIV), hematopoieticneoplasias, multiple myeloma, Castleman's disease or cardiac myxoma.

In addition, the compounds may be incorporated into biodegradablepolymers allowing for sustained release, the polymers being implanted inthe vicinity of where delivery is desired, for example, at the site of abacterial infection. The biodegradable polymers and their use aredescribed in detail in Brem et al., J. Neurosurg, 74:441-446 (1991).

As mentioned above, the present invention provides a pharmaceuticalformulation having an effective amount of a compound of formula I fortreating a patient having a bacterial infection. As used herein, aneffective amount of the compound is defined as the amount of thecompound which, upon administration to a patient, inhibits growth ofbacteria, kills bacteria cells, sensitizes bacteria to otherantibiotics, or eliminates the bacterial infection entirely in thetreated patient. The dosage of the composition will depend on thecondition being treated, the particular derivative used, and otherclinical factors such as weight and condition of the patient and theroute of administration of the compound. However, for oraladministration to humans, a dosage of 0.01 to 100 mg/kg/day, preferably0.01-1 mg/kg/day, is generally sufficient. Effective doses will alsovary, as recognized by those skilled in the art, dependent on route ofadministration, excipient usage, and the possibility of co-usage withother therapeutic treatments including other antibiotic agents.

For example, the term “therapeutically effective amount” as used hereinfor treatment of endotoxemia refers to the amount of compound used is ofsufficient quantity to decrease the subject's response to LPS anddecrease the symptoms of sepsis. The term “therapeutically effective”therefore includes that the amount of compound sufficient to prevent,and preferably reduce by at least 50%, and more preferably sufficient toreduce by 90%, a clinically significant increase in the plasma level ofTNF. The dosage ranges for the administration of compound are thoselarge enough to produce the desired effect. Generally, the dosage willvary with the age, condition, sex, and extent of the infection withbacteria or other agent as described above, in the patient and can bedetermined by one skilled in the art. The dosage can be adjusted by theindividual physician in the event of any contraindications. In anyevent, the effectiveness of treatment can be determined by monitoringthe level of LPS and TNF in a patient. A decrease in serum LPS and TNFlevels should correlate with recovery of the patient.

In addition, patients at risk for or exhibiting the symptoms of sepsiscan be treated by the method as described above, further comprisingadministering, substantially simultaneously with the therapeuticadministration of compound, an inhibitor of TNF, an antibiotic, or both.For example, intervention in the role of TNF in sepsis, either directlyor indirectly, such as by use of an anti-TNF antibody and/or a TNFantagonist, can prevent or ameliorate the symptoms of sepsis.Particularly preferred is the use of an anti-TNF antibody as an activeingredient, such as a monoclonal antibody with TNF specificity asdescribed by Tracey, et al. (Nature, 330:662, 1987).

A patient who exhibits the symptoms of sepsis may be treated with anantibiotic in addition to the treatment with compound. Typicalantibiotics include an aminoglycoside, such as gentamicin or abeta-lactam such as penicillin, or cephalosporin or any of theantibiotics as previously listed above. Therefore, a preferredtherapeutic method of the invention includes administering atherapeutically effective amount of cationic compound substantiallysimultaneously with administration of a bactericidal amount of anantibiotic. Preferably, administration of compound occurs within about48 hours and preferably within about 2-8 hours, and most preferably,substantially concurrently with administration of the antibiotic.

The term “bactericidal amount” as used herein refers to an amountsufficient to achieve a bacteria-killing blood concentration in thepatient receiving the treatment. The bactericidal amount of antibioticgenerally recognized as safe for administration to a human is well knownin the art, and as is known in the art, varies with the specificantibiotic and the type of bacterial infection being treated.

Because of the antibiotic, antimicrobial, and antiviral properties ofthe compounds, they may also be used as preservatives or sterillants ofmaterials susceptible to microbial or viral contamination. The compoundsof the invention can be utilized as broad-spectrum antimicrobial agentsdirected toward various specific applications. Such applications includeuse of the compounds as preservatives in processed foods when verifiedas effective against organisms including Salmonella, Yersinia, Shigella,either alone or in combination with antibacterial food additives such aslysozymes; as a topical agent (Pseudomonas, Streptococcus) and to killodor producing microbes (Micrococci). The relative effectiveness of thecompounds of the invention for the applications described can be readilydetermined by one of skill in the art by determining the sensitivity ofany organism to one of the compounds.

While primarily targeted at classical Gram-negative-staining bacteriawhose outer capsule contains a substantial amount of lipid A, it mayalso be effective against other organisms with a hydrophobic outercapsule. For example, Mycobacterium spp. have a waxy protective outercoating, and compounds of the invention in combination with antibioticsmay provide enhanced effectiveness against Mycobacterial infection,including tuberculosis. In that case, the compounds could beadministered nasally (aspiration), by any of several known techniques.

Apart from anti-microbial action, the permeability provided by thecompounds may enhance introduction of a great variety of substances intomicrobes. For example, the compounds may be used to enhance introductionof macromolecules such as DNA or RNA into microbes, particularlyGram-negative bacteria. In that case, there may be no need for thetraditional vectors (e.g., phages) used to package nucleic acids whentransfecting the microbes. Conditions and techniques for introducingsuch macromolecules into microbes using the compounds of the inventionwill in most cases be routine.

The formulations include those suitable for oral, rectal, nasal, topical(including buccal and sublingual), vaginal or parenteral (includingsubcutaneous, intramuscular, intravenous, intradermal, intraocular,intratracheal, and epidural) administration. The formulations mayconveniently be presented in unit dosage form and may be prepared byconventional pharmaceutical techniques. Such techniques include the stepof bringing into association the active ingredient and thepharmaceutical carrier(s) or excipient(s). In general, the formulationsare prepared by uniformly and intimately bringing into associate theactive ingredient with liquid carriers or finely divided solid carriersor both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil emulsion and as a bolus, etc.

A tablet may be made by compression or molding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing, in a suitable machine, the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, preservative, surface-active ordispersing agent. Molded tablets may be made by molding, in a suitablemachine, a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide a slow or controlled release of theactive ingredient therein.

Formulations suitable for topical administration in the mouth includelozenges comprising the ingredients in a flavored basis, usually sucroseand acacia or tragacanth; pastilles comprising the active ingredient inan inert basis such as gelatin and glycerin, or sucrose and acacia; andmouthwashes comprising the ingredient to be administered in a suitableliquid carrier.

Formulations suitable for topical administration to the skin may bepresented as ointments, creams, gels and pastes comprising theingredient to be administered in a pharmaceutical acceptable carrier. Apreferred topical delivery system is a transdermal patch containing theingredient to be administered.

Formulations for rectal administration may be presented as a suppositorywith a suitable base comprising, for example, cocoa butter or asalicylate.

Formulations suitable for nasal administration, wherein the carrier is asolid, include a coarse powder having a particle size, for example, inthe range of 20 to 500 microns which is administered in the manner inwhich snuff is taken, i.e., by rapid inhalation through the nasalpassage from a container of the powder held close up to the nose.Suitable formulations, wherein the carrier is a liquid, foradministration, as for example, a nasal spray or as nasal drops, includeaqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or spray formulationscontaining in addition to the active ingredient such as carriers as areknown in the art to be appropriate.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, other bacteriostats and solutes which render the formulationisotonic with the blood of the intended recipient; and aqueous andnon-aqueous sterile suspensions which may include suspending agents andthickening agents. The formulations may be presented in unit-dose ormulti-dose containers, for example, sealed ampules and vials, and may bestored in a freeze-dried (lyophilized) conditions requiring only theaddition of the sterile liquid carrier, for example, water forinjections, immediately prior to use. Extemporaneous injection solutionsand suspensions may be prepared from sterile powders, granules andtables of the kind previously described.

Preferred unit dosage formulations are those containing a daily dose orunit, daily sub-dose, as herein above recited, or an appropriatefraction thereof, of the administered ingredient.

It should be understood that in addition to the ingredients,particularly mentioned above, the formulations of this invention mayinclude other agents conventional in the art having regard to the typeof formulation in question, for example, those suitable for oraladministration may include flavoring agents.

The carrier in the pharmaceutical composition must be “acceptable” inthe sense of being compatible with the active ingredient of theformulation (and preferably, capable of stabilizing it) and notdeleterious to the subject to be treated.

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following specificembodiments are, therefore, to be construed as merely illustrative, andnot limitative of the remainder of the disclosure in any way whatsoever.All of the publications cited herein, including patents, are herebyincorporated by reference.

Examples 1-14 represent typical syntheses of compounds 1 through 343,some of which are shown in Schemes 1 through 16. Example 14 showsstability of compounds 352-354 under acidic, neutral and basicconditions. Example 15 represents other compounds of formula I which canbe synthesized using known starting materials and reaction schemes thatare similar to those described herein. For example, the hydroxyl groupson cholic acid can be converted into amine groups by the method found inHsieh et al., Synthesis and DNA Binding Properties of C3-, C12-, andC24-Substituted Amino-Steroids Derived from Bile Acids, Biorganic andMedicinal Chemistry, 1995, vol. 6, 823-838. Example 16 represents MICand MCB testing, and Example 17 represents the ability of the compoundsof formula I to lower the MIC's of other antibiotics.

EXAMPLES

The examples illustrate particular synthesis of some particularcompounds useful in the methods described herein. For example,representative syntheses of some of the compounds 1-343 are presentedbelow.

¹H and ¹³C NMR spectra were recorded on a Varian Gemini 2000 (200 MHz),Varian Unity 300 (300 MHz), or Varian VXR 500 (500 MHz) spectrometer andare referenced to TMS, residual CHCl₃ (¹H) or CDCl₃ (¹³C), or residualCHD₂OD (¹H), or CD₃OD (¹³C). IR spectra were recorded on a Perkin Elmer1600 FTIR instrument. Mass spectrometric data were obtained on a JOEL SX102A spectrometer. THF was dried over Na/benzophenone and CH₂Cl₂ wasdried over CaH₂ prior to use. Other reagents and solvents were obtainedcommercially and were used as received.

Example 1 Syntheses of Compounds 1, 2, 4, 5, 13-20 and 22-27

Compound 13: To a 1 L round-bottom flask were added methyl cholate(30.67 g, 72.7 mmol) in dry THF (600 mL) and LiAlH₄ (4.13 g, 109 mmol).After reflux for 48 hours, saturated aqueous Na₂SO₄(100 mL) wasintroduced slowly, and the resulted precipitate was filtered out andwashed with hot THF and MeOH. Recrystallization from MeOH gave colorlesscrystals of 13 (28.0 g, 98% yield). m.p. 236.5-238° C.; IR (KBr) 3375,2934, 1373, 1081 cm⁻¹; ¹H NMR (CDCl₃/MeOH-d4, 200 MHz) δ 3.98 (bs, 1 H),3.83 (bs, 1 H), 3.60-3.46 (m, 2 H), 3.38 (bs, 5 H), 2.30-2.10 (m, 2 H),2.05-1.05 (series of multiplets, 22 H), 1.03 (bs, 3 H), 0.92 (s, 3 H),0.71 (s, 3 H); ¹³C NMR (CDCl₃/MeOH-d4, 50 MHz) δ 73.89, 72.44, 68.99,63.51, 48.05, 47.12, 42.49, 40.37, 39.99, 36.62, 36.12, 35.58, 35.40,32.77, 30.69, 30.04, 29.02, 28.43, 27.27, 23.96, 23.08, 18.00, 13.02;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 417.2992 (55.3%);calcd. 417.2981.

Compound 14: To a round-bottom flask were added 13 (28.2 g, 71.7 mmol)in DMF (300 ml), Et₃N (20 mL, 143.4 mmol), trityl chloride (25.98 g,93.2 mmol) and DMAP (0.13 g, 1.07 mmol). The mixture was stirred at 50°C. under N₂ for 30 hours followed by the introduction of water (1000 mL)and extraction with EtOAc (5×200 mL). The combined extracts were washedwith water and brine and then dried over MgSO₄. After removal of solventin vacuo, the residue was purified using SiO₂ chromatography (CH₂Cl₂,Et₂O and MeOH as eluents) to give 14 as a pale yellow solid (31.9 g, 70%yield). m.p. 187° C. (decomposition); IR (KBr) 3405, 2935, 1448, 1075cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.46-7.42 (m, 6 H), 7.32-7.17 (m, 9 H),3.97 (bs, 1 H), 3.83 (bs, 1 H), 3.50-3.38 (m, 1 H), 3.01 (bs, 1 H), 2.94(dd, J=14.2, 12.2 Hz, 2 H), 2.64 (bs, 1 H), 2.51 (bs, 1 H), 2.36-2.10(m, 2 H), 2.00-1.05 (series of multiplets, 22 H), 0.96 (d, J=5.8 Hz, 3H), 0.87 (s, 3 H), 0.64 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz)-144.77,128.93, 127.91, 127.01, 86.43, 73.35, 72.06, 68.66, 64.28, 47.47, 46.53,41.74, 41.62, 39.64, 35.57, 35.46, 34.91, 34.82, 32.40, 30.55, 28.21,27.69, 26.80, 26.45, 23.36, 22.59, 17.83, 12.61; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 659.4069 (100%); calcd.659.4076.

Compound 15: To a round-bottom flask were added 14 (20.0 g, 31.4 mmol)in dry THF (600 mL) and NaH (60% in mineral oil, 6.3 g, 157.2 mmol). Themixture was refluxed for 30 min under N₂ followed by addition of allylbromide (27 mL, 314 mmol). After 60 hours of reflux, additional NaH (3eq.) and allyl bromide (4 eq.) were added. Following another 50 hours ofreflux, water (20 mL) was introduced slowly followed by addition of 1%HCl until the aqueous layer became neutral. The mixture was thenextracted with ether (3×100 mL) and the combined extracts were washedwith water (100 mL) and brine (2×100 mL). The ether solution was driedover anhydrous Na₂SO₄, and after removal of solvent, the residue waspurified using SiO₂ chromatography (hexanes and EtOAc/hexanes 1:8 aseluents) to give 15 (22.76 g, 96% yield) as a pale yellow glass. IR(neat) 2930, 1448, 1087 cm⁻¹; ¹H NMR (CDCl₃, 200 M Hz) δ 7.48-7.30 (m, 6H), 7.32-7.14 (m, 9 H), 6.04-5.80 (m, 3 H), 5.36-5.04 (series ofmultiplets, 6 H), 4.14-3.94 (m, 4 H), 3.74 (td, J=13.8, 5.8 Hz, 2 H),3.53 (bs, 1 H), 3.20-2.94 (m, 3 H), 3.31 (bs, 1 H), 2.38-1.90 (m, 4 H),1.90-0.96 (series of multiplets, 20 H), 0.90 (d, J=5.4 Hz, 3 H), 0.89(s, 3 H), 0.64 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.83, 136.27,136.08, 128.94, 127.90, 126.98, 116.46, 115.70, 86.42, 80.94, 79.29,74.98, 69.52, 69.39, 68.86, 64.39, 46.51, 46.42, 42.67, 42.14, 39.92,35.63, 35.51, 35.13, 32.45, 28.98, 28.09, 27.66, 27.57, 26.72, 23.32,23.11, 17.92, 12.69; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)779.5013 (86.1%); calcd. 779.5015.

Compound 16: To a three-necked round bottom flask was added 15 (3.34 g,4.4 mmol) in CH₂Cl₂ (200 mL) and methanol (100 mL). Through the coldsolution (−78° C.) ozone was bubbled through until a blue colorpersisted. Excess ozone was removed with oxygen flow. The mixture wasleft in a dry ice-acetone bath for an hour. Methyl sulfide (2.4 mL) wasadded and 15 minutes later, the mixture was treated with NaBH₄ (1.21 g,32 mmol) in 5% aqueous NaOH solution (10 mL)/methanol (10 mL) andallowed to warm to room temperature. The mixture was washed with brine(3×50 mL), and the combined brine wash was extracted with CH₂Cl₂ (2×50mL). The organic solution was dried over MgSO₄. After SiO₂chromatography (MeOH (5%) in CH₂Cl₂), 3.30 g (95% yield) of 16 wasisolated as an oil. IR (neat) 3358, 2934, 1448, 1070 cm⁻¹; ¹H NMR(CDCl₃, 200 MHz) a 7.50-7.42 (m, 6 H), 7.32-7.17 (m, 9 H), 3.80-2.96(series of multiplets, 20 H), 2.25-0.96 (series of multiplets, 24 H),0.89 (bs, 6 H), 0.65 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 144.73, 128.88,127.87, 126.96, 86.38, 81.05, 79.75, 76.59, 70.33, 69.66, 69.30, 64.20,62.25, 62.16, 62.03, 46.77, 46.36, 42.63, 41.77, 39.60, 35.43, 35.23,35.05, 34.89, 32.42, 28.91, 27.93, 27.56, 27.15, 26.68, 23.35, 22.98,22.85, 18.15, 12.60; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)791.4860 (100%), calcd. 791.4863.

Compound 17: To a round-bottom flask was added 16 (1.17 g, 1.55 mmol) indry THF (30 mL) under N₂ in ice-bath followed by 9-BBN/THF solution (0.5M, 10.2 mL, 5.51 mmol). The mixture was stirred at room temperature for12 hours. Aqueous NaOH (20%) (2 mL) and hydrogen peroxide (30%) (2 mL)were added in sequence. The mixture was refluxed for 1 hour followed bythe addition of brine (60 mL) and extraction with EtOAc (4×30 mL). Thecombined extracts were dried over anhydrous Na₂SO₄. The product (1.01 g,80% yield) was obtained as a colorless-oil after SiO₂ chromatography (5%MeOH in CH₂Cl₂). IR (neat) 3396, 2936, 1448, 1365, 1089 cm⁻¹; ¹HNMR(CDCl₃, 200 MHz) δ 7.50-7.42 (m, 6 H), 7.34-7.16 (m, 9 H), 3.90-3.56(m, 13H), 3.50 (bs, 1 H), 3.40-2.96 (series of multiplets, 6 H),2.30-0.94 (series of multiplets, 30H), 0.90 (s, 3 H), 0.88 (d, J=5.4 Hz,3 H), 0.64 (s, 3 H); ¹³C NMR(CDCl₃, 50 MHz) δ 144.73, 128.88, 127.85,126.94, 86.36, 80.52, 78.90, 76.36, 66.82, 66.18, 65.77, 64.22, 61.53,61.41, 61.34, 46.89, 46.04, 42.60, 41.59, 39.60, 35.37, 35.27, 34.88,32.75, 32.44, 32.31, 28.82, 27.65, 27.48, 27.13, 26.77, 23.35, 22.74,22.38, 18.08, 12.48; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)833.5331 (100%), calcd. 833.5332.

Compound 18: To a round-bottom flask were added 16 (3.30 g, 4.29 mmol)in CH₂Cl₂ (150 mL) and NEt₃(2.09 mL, 15.01 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (1.10 mL, 14.16mmol). After 30 minutes, water (30 mL) and brine (200 mL) were added.The CH₂Cl₂ layer was washed with brine (2×50 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×100 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (3.35 g, 78% yield) was isolatedas a pale yellow oil after SiO₂ chromatography (EtOAc/hexanes 1:1). IR(neat) 2937, 1448, 1352, 1174, 1120, 924 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ7.52-7.40 (m, 6 H), 7.34-7.20, (m, 9 H), 4.42-4.24 (m, 6 H), 3.90-3.64(m, 4 H), 3.60-3.30 (m, 4 H), 3.24-3.00 (m, 3 H), 3.10 (s, 6 H), 3.05(s, 3 H), 2.20-1.96 (m, 3 H)1.96-1.60 (m, 8 H), 1.60-0.94 (series ofmultiplets, 13 H), 0.91 (bs, 6 H), 0.65 (s, 3 H); ¹³C NMR(CDCl₃, 50 MHz)δ 114.68, 128.85, 127.85, 126.96, 86.37, 81.37, 79.58, 76.58, 69.95,69.43, 69.34, 66.52, 66.31, 65.59, 64.11, 46.80, 46.20, 42.65, 41.48,39.35, 37.82, 37.48, 35.36, 34.92, 34.73, 32.37, 28.66, 28.01, 27.44,27.03, 26.72, 23.17, 22.91, 22.72, 18.13, 12.50; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 1205.4176 (81.5%), calcd.1205.4189.

Compound 19: To a round-bottom flask were added 17 (1.01 g, 1.25 mmol)in CH₂Cl₂ (50 mL) and NEt₃ (0.608 mL, 4.36 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.318 mL, 4.11mmol). After 30 minutes, water (10 mL) and then brine (80 mL) wereadded. The CH₂Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×40 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (1.07 g, 82%) was isolated as apale yellowish oil after SiO₂ chromatography (EtOAc/hexanes 1:1). IR(neat) 2938, 1356, 1176, 1112 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.43,(m, 6 H), 7.32-7.22 (m, 9 H), 4.40-4.31 (m, 6 H), 3.72-3.64 (m, 2 H),3.55 (dd, J=6.3, 5.8 Hz, 2H), 3.51 (bs, 1 H), 3.32-3.14 (m, 3 H),3.14-2.92 (m, 3 H), 3.01 (s, 3 H), 3.01 (s, 3 H), 3.00 (s, 3 H),2.10-1.92 (m, 10 H), 1.92-1.58 (m, 8 H), 1.56-0.92 (series ofmultiplets, 12H), 0.90 (s, 3 H), 0.89 (d, J=5.4 Hz, 3 H), 0.64 (s, 3 H);¹³C NMR(CDCl₃, 75 MHz) δ 144.67, 128.85, 127.85, 126.96, 86.42, 81.06,79.83, 76.81, 68.12, 68.06, 68.02, 64.26, 64.06, 63.42, 46.76, 46.38,42.73, 41.87, 39.73, 37.44, 37.32, 37.29, 35.52, 35.48, 35.32, 35.06,32.53, 30.55, 30.28, 30.02, 29.15, 27.96, 27.69, 27.61, 26.75, 23.52,23.02, 18.17, 12.64; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)1067.4672 (100%), calcd. 1067.4659.

Compound 20: To a round-bottom flask were added 18 (1.50 g, 1.50 mmol)in dry DMSO (20 mL) and NaN₃ (0.976 g, 15 mmol). The mixture was heatedto 80° C. and stirred under N₂ overnight then diluted with water (100mL). The resulted aqueous mixture was extracted with EtOAc (3×50 mL),and the combined extracts washed with brine and dried over anhydrousNa₂SO₄. The desired product (0.83 g, 66% yield) was isolated as a clearglass after SiO₂ chromatography (EtOAc/hexanes 1:5). IR (neat) 2935,2106, 1448, 1302, 1114 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 7.50-7.42 (m, 6H), 7.36-7.20 (m, 9 H), 3.84-3.70 (m, 2 H), 3.65 (t, J=4.9 Hz, 2 H),3.55 (bs, 1 H), 3.44-3.08 (m, 10 H), 3.02 (t, J=6.4 Hz, 2 H), 2.38-0.96(series of multiplets, 24 H), 0.92 (d, J=5.6 Hz, 3 H), 0.91 (s, 3 H),0.65 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 114.84, 128.97, 127.92, 126.99,86.42, 81.24, 80.12, 76.59, 67.84, 67.29, 66.66, 64.36, 51.67, 51.44,51.18, 46.53, 46.23, 42.21, 41.93, 39.73, 35.66, 35.36, 35.06, 34.78,32.40, 28.95, 27.76, 27.39, 26.87, 23.45, 22.98, 22.92, 17.98, 12.53;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 866.5040 (100%),calcd. 866.5057.

Compound 22: To a round-bottom flask were added 20 (830 mg, 0.984 mmol)in MeOH (30 mL) and CH₂Cl₂ (30 mL) and p-toluenesulfonic acid (9.35 mg,0.0492 mmol). The solution was stirred at room temperature for 2.5 hoursthen saturated aqueous NaHCO₃ (10 mL) was introduced. Brine (30 mL) wasadded, and the mixture was extracted with EtOAc (4×20 mL). The combinedextracts were dried over anhydrous Na₂SO₄. The desired product (0.564 g,95% yield) was isolated as a pale yellowish oil after SiO₂chromatography (EtOAc/hexanes 1:2). IR (neat) 3410, 2934, 2106, 1301,1112 cm⁻¹; ¹H NMR (CDCl₃, 200 MHz) δ 3.80-3.54 (m, 7 H), 3.44-3.20 (m,10 H), 2.35-0.96 (series of multiplets, 24 H), 0.95 (d, J=6.4 Hz, 3 H),0.92 (s, 3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 81.10, 80.01,76.60, 67.75, 67.16, 66.56, 63.63, 51.57, 51.34, 51.06, 46.29, 46.12,42.12, 41.81, 39.60, 35.55, 35.23, 34.94, 34.66, 31.75, 29.48, 28.81,27.72, 27.66, 27.29, 23.32, 22.86, 22.80, 17.85, 12.39; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 624.3965 (100%), calcd.624.3962.

Compound 23: To a round-bottom flask were added 19 (1.07 g, 1.025 mmol)and NaN₃ (0.666 g, 10.25 mmol) followed the introduction of dry DMSO (15mL). The mixture was heated up to 80° C. under N₂ overnight. After theaddition of H₂O (100 mL), the mixture was extracted with EtOAc (4×40 mL)and the combined extracts were washed with brine (2×50 mL) and driedover anhydrous Na₂SO₄. After removal of solvent, the residue wasdissolved in MeOH (15 mL) and CH₂Cl₂ (15 mL) followed by the addition ofcatalytic amount of p-toluenesulfonic acid (9.75 mg, 0.051 mmol). Thesolution was stirred at room temperature for 2.5 hours before theaddition of saturated NaHCO₃ solution (15 mL). After the addition ofbrine (60 mL), the mixture was extracted with EtOAc (5×30 mL). Thecombined extracts were washed with brine (50 mL) and dried overanhydrous Na₂SO₄. The desired product (0.617 g, 94% yield for two steps)was obtained as a yellowish oil after SiO₂ chromatography (EtOAc/hexanes1:2). IR (neat) 3426, 2928, 2094, 1456, 1263, 1107 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ 3.68-3.56 (m, 3 H), 3.56-3.34 (series of multiplets, 10 H),3.28-3.00 (series of multiplets, 4 H), 2.20-2.00 (m, 3 H), 1.98-1.55(series of multiplets, 15 H), 1.55-0.96 (series of multiplets, 13 H),0.92 (d, J=6.6 Hz, 3 H), 0.89 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃,75 MHz) δ 80.63, 79.79, 76.04, 64.99, 64.45, 64.30, 63.72, 49.01, 48.94,48.74, 46.49, 46.39, 42.70, 41.98, 39.80, 35.65, 35.42, 35.28, 35.08,31.99, 29.78, 29.75, 29.70, 29.49, 29.06, 27.87, 27.79, 27.65, 23.53,23.04, 22.85, 18.05, 12.59; HRFAB-MS (thioglycerol+Na matrix) m/e:([M+Na]⁺) 666.4415 (100%), calcd. 666.4431.

Compound 24: To a round-bottom flask were added 22 (0.564 g, 0.938 mmol)in CH₂Cl₂ (30 mL) and NEt₃ (0.20 mL, 1.40 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.087 mL, 1.13mmol). After 30 minutes, water (20 mL) and brine (100 mL) were added.The CH₂CL₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×30 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (0.634 g, 99% yield) was isolatedas a pale yellowish oil after SiO₂ chromatography (EtOAc/hexanes 1:2).IR (neat) 2935, 2106, 1356, 1175, 1113 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ4.20 (t, J=6.8 Hz, 2 H), 3.80-3.75 (m, 1 H), 3.70-3.64 (m, 3 H), 3.55(bs, 1 H), 3.44-3.01 (m, 10 H), 3.00 (s, 3 H), 2.32-2.17 (m, 3 H),2.06-2.03 (m, 1 H), 1.90-0.88 (series of multiplets, 20 H), 0.95 (d,J=6.6 Hz, 3 H), 0.91 (s, 3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ80.90, 79.86, 76.43, 70.78, 67.64, 66.99, 66.48, 51.50, 51.26, 50.97,46.05, 45.96, 42.08, 41.71, 39.51, 37.33, 35.15, 34.86, 34.60, 31.34,28.73, 27.62, 27.59, 27.51, 25.68, 23.22, 22.80, 22.70, 17.62, 12.33;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 702.3741 (100%),calcd. 702.3737.

Compound 25: To a round-bottom flask were added 23 (0.617 g, 0.96 mmol)in CH₂Cl₂ (30 mL) and NEt₃ (0.20 mL, 1.44 mmol). The mixture was put inice-bath under N₂ followed by addition of mesyl chloride (0.089 mL, 1.15mmol). After 30 minutes, water (20 mL) and brine (120 mL) were added.The CH₂Cl₂ layer was washed with brine (2×20 mL) and dried overanhydrous Na₂SO₄. The combined aqueous mixture was extracted with EtOAc(3×30 mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The desired product (0.676 g, 97% yield) was isolatedas a pale yellowish oil after removal of solvent. IR (neat) 2934, 2094,1454, 1360, 1174, 1112 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 4.17 (t, J=6.6Hz, 2 H), 3.65-3.28 (series of multiplets, 11 H), 3.64-3.00 (series ofmultiplets, 4 H), 2.97 (s, 3 H), 2.18-1.96 (series of multiplets, 16 H),1.54-0.94 (series of multiplets, 11 H), 0.89 (d, J=6.6 Hz, 3 H), 0.86(s, 3 H), 0.63 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.47, 79.67, 75.92,70.84, 64.90, 64.37, 64.17, 48.90, 48.86, 48.66, 46.32, 46.26, 42.63,41.87, 39.70, 37.39, 35.34, 35.28, 35.20, 34.99, 31.61, 29.68, 29.60,28.96, 27.78, 27.68, 27.57, 25.79, 23.41, 22.95, 22.74, 17.82, 12.50;HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺) 722.4385 (22.1%), calcd.722.4387.

Compound 26: To a 50 mL round-bottom flask was added 24 (0.634 g, 0.936mmol) and N-benzylmethylamine (2 mL). The mixture was heated under N₂ at80° C. overnight. Excess N-benzylmethylamine was removed under vacuum,and the residue was subjected to SiO₂ chromatography (EtOAc/hexanes1:2). The desired product (0.6236 g, 95% yield) was isolated as a paleyellow oil. IR (neat) 2935, 2106, 1452, 1302, 1116 cm⁻¹; ¹H NMR (CDCl₃,200 MHz) δ 7.32-7.24 (m, 5 H), 3.80-3.76 (m, 1 H), 3.70-3.60 (m, 3 H),3.54 (bs, 1 H), 3.47 (s, 2 H), 3.42-3.10 (m, 10 H), 2.38-2.05 (m, 5 H),2.17 (s, 3 H), 2.02-0.88 (series of multiplet, 21 H), 0.93 (d, J=7.0 Hz,3 H), 0.91 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 139.60,129.34, 128.38, 127.02, 81.22, 80.10, 76.71, 67.85, 67.29, 66.65, 62.45,58.38, 51.65, 51.44, 51.16, 46.50, 46.21, 42.40, 42.20, 41.93, 39.72,35.80, 35.34, 35.05, 34.76, 33.65, 28.93, 27082, 27.75, 27.38, 24.10,23.45, 22.98, 22.91, 18.05, 12.50; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M−H]⁺) 703.4748 (90.2%), calcd. 703.4772; ([M+H]⁺) 705.4911(100%), calcd. 705.4928; ([M+Na]⁺) 727.4767 (1.5%), calcd. 727.4748.

Compound 27: To a 50 mL round-bottom flask was added 25 (0.676 g, 0.937mmol) and N-benzylmethylamine (2 mL). The mixture was heated under N₂ at80° C. overnight. Excess N-benzylmethylamine was removed under vacuumand the residue was subjected to SiO₂ chromatography (EtOAc/hexanes1:2). The desired product (0.672 g, 96% yield) was isolated as a paleyellow oil. IR (neat) 2934, 2096, 1452, 1283, 1107 cm⁻¹; ¹H NMR (CDCl₃,300 MHz) δ 7.34-7.20 (m, 5 H), 3.68-3.37 (series of multiplets, 13 H),3.28-3.04 (m, 4 H), 2.33 (t, J=7.0 Hz, 2 H), 2.18 (s, 3 H), 2.20-2.00(m, 3 H), 1.96-1.56 (series of multiplets, 14 H), 1.54-1.12 (m, 10 H),1.10-0.96 (m, 3 H), 0.91 (d, J=8.7 Hz, 3 H), 0.89 (s, 3 H), 0.65 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.48, 129.23, 128.30, 126.96, 80.66,79.81, 76.08, 65.00, 64.46, 64.34, 62.50, 58.37, 49.02, 48.95, 48.75,46.65, 46.40, 42.69, 42.43, 42.00, 39.83, 35.86, 35.45, 35.30, 35.10,33.83, 29.81, 29.78, 29.72, 29.09, 27.88, 27.81, 27.66, 24.19, 23.57,23.06, 22.87, 18.15, 12.62; HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺)747.5406 (77.2%), calcd. 747.5398.

Compound 1: To a round-bottom flask were added 26 (0.684 g, 0.971 mmol)in dry THF (30 mL) and LiAlH₄ (113.7 mg, 3.0 mmol) under N₂. The mixturewas stirred at room temperature for 12 hours, and then Na₂SO₄.10H₂Opowder (10 g) was added slowly. After the grey color disappeared, themixture was filtered through Celite and washed with dry THF. The product(0.581 g, 95% yield) was obtained as a colorless glass. IR (neat) 3372,2937, 1558, 1455, 1362, 1102 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.20(m, 5 H), 3.68-3.48 (m, 5 H), 3.47 (s, 2 H), 3.29 (bs, 1 H), 3.22-3.00(m, 3 H), 2.96-2.80 (m, 6 H), 2.32 (t, J=6.8, 5.4 Hz, 2 H), 2.17 (s, 3H), 2.20-2.00 (m, 3 H), 1.96-0.96 (series of multiplets, 27 H), 0.93 (d,J=6.8 Hz, 3 H), 0.90, (s, 3 H), 0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ139.50, 129.22, 128.31, 126.96, 80.76, 79.85, 76.10, 70.90, 70.33,70.24, 62.48, 58.27, 46.55, 46.45, 42.72, 42.58, 42.33, 41.99, 39.77,35.78, 35.37, 35.01, 33.73, 29.07, 27.95, 27.71, 24.06, 23.46, 22.99,18.14, 12.55; HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺) 627.5211(100%), calcd. 627.5213.

HCl salt of compound 1: Compound 1 was dissolved in a minimum amount ofCH₂Cl₂ and excess HCl in ether was added. Solvent and excess HCl wereremoved in vacuo and a noncrystalline white powder was obtained. ¹H NMR(methanol-d4/15% CDCl₃, 300 MHz) δ 7.61-7.57 (m, 2 H), 7.50-7.48 (m, 3H), 4.84 (bs, 10 H), 4.45 (bs, 1H), 4.30 (bs, 1 H), 3.96-3.82 (m, 2 H),3.78-3.69 (m, 2 H), 3.66 (bs, 1 H), 3.59-3.32 (series of multiplets, 4H), 3.28-3.02 (m, 8 H), 2.81 (s, 3 H), 2.36-2.15 (m, 4 H), 2.02-1.68 (m,8 H), 1.64-0.90 (series of multiplets, 12 H), 1.01 (d, J=6.35 Hz, 3 H),0.96 (s, 3 H), 0.73 (s, 3 H); ¹³C NMR (methanol-d4/15% CDCl₃, 75 MHz) δ132.31, 131.20, 130.92, 130.40, 83.13, 81.09, 78.48, 65.54, 64.98,64.11, 60.87, 57.66, 47.51, 46.91, 43.52, 43.00, 41.38, 41.19, 41.16,40.75, 40.30, 36.37, 36.08, 36.00, 35.96, 33.77, 29.68, 29.34, 28.65,28.37, 24.42, 24.25, 23.33, 21.51, 18.80, 13.04.

Compound 2: To a round-bottom flask were added 27 (0.82 g, 1.10 mmol) indry THF (150 mL) and LiAlH₄ (125 mg, 3.30 mmol) under N₂. The mixturewas stirred at room temperature for 12 hours and Na₂SO₄.10H₂O powder (10g) was added slowly. After the grey color disappeared, the mixture wasfiltered through a cotton plug and washed with dry THF. THF was removedin vacuo and the residue dissolved in CH₂Cl₂ (50 mL). After filtration,the desired product was obtained as a colorless glass (0.73 g, 99%yield). IR (neat) 3362, 2936, 2862, 2786, 1576, 1466, 1363, 1103 cm⁻¹;¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.23 (m, 5 H), 3.67-3.63 (m, 1 H),3.60-3.57 (m, 1 H), 3.53 (t, J=6.4 Hz, 2 H), 3.47 (s, 2 H), 3.46 (bs, 1H), 3.24-3.17(m, 2 H), 3.12-2.99 (m, 2 H), 2.83-2.74 (series ofmultiplets, 6 H), 2.30 (t, J=7.3 Hz, 2 H), 2.15 (s, 3 H), 2.20-2.00 (m,3 H), 1.95-1.51 (series of multiplets, 20 H), 1.51-1.08, (series ofmultiplets, 10 H), 1.06-0.80 (m, 3 H), 0.87 (d, J=8.1 Hz, 3 H), 0.86 (s,3 H), 0.61 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.35, 129.16, 128.22,126.88, 80.44, 79.29, 75.96, 66.70, 66.52, 66.12, 62.45, 58.26, 46.76,46.27, 42.69, 42.41, 42.02, 40.68, 40.10, 40.02, 39.82, 35.84, 35.47,35.30, 35.06, 34.15, 34.09, 34.03, 33.80, 28.96, 27.93, 27.75, 27.71,24.32, 23.53, 23.03, 22.75, 18.17, 12.58; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 691.5504 (38.5%), calcd. 691.5502.

HCl salt of compound 2: Compound 2 was dissolved in a minimum amount ofCH₂Cl₂ and excess HCl in ether was added. Removal of the solvent andexcess HCl gave a noncrystalline white powder. ¹H NMR (methanol-d4/15%CDCl₃, 300 MHz) δ 7.60-7.59(m, 2 H), 7.50-7.47 (m, 3 H), 4.82 (bs, 10H), 4.43 (bs, 1 H), 4.32 (bs, 1 H), 3.85-3.79 (m, 1 H), 3.75-3.68 (m, 1H), 3.64 (t, J=5.74 Hz, 2 H), 3.57 (bs, 1 H), 3.36-3.28 (m, 2 H),3.25-3.00 (series of multiplets, 10 H), 2.82 (s, 3 H), 2.14-1.68 (seriesof multiplets, 19 H), 1.65-1.15 (series of multiplets, 11 H), 0.98 (d,J=6.6 Hz, 3 H), 0.95 (s, 3 H), 0.72 (s, 3 H); ¹³C NMR (methanol-d4/15%CDCl₃, 75 MHz) δ 132.21, 131.10, 130.58, 130.28, 81.96, 80.72, 77.60,66.84, 66.58, 66.12, 61.03, 57.60, 44.16, 42.77, 40.62, 39.57, 39.43,36.28, 36.03, 35.96, 35.78, 33.65, 29.48, 29.27, 29.11, 29.01, 28.61,28.56, 28.35, 24.25, 23.56, 23.30, 21.17, 18.64, 12.90.

Compound 4: A suspension of 1 (79.1 mg, 0.126 mmol) andaminoiminomethanesulfonic acid (50.15 mg, 0.404 mmol) in methanol andchloroform was stirred at room temperature for 24 hours, and thesuspension became clear. An ether solution of HCl (1 M, 1 mL) was addedfollowed by the removal of solvent with N₂ flow. The residue wasdissolved in H₂O (5 mL) followed by the addition of 20% aqueous NaOH(0.5 mL). The resulting cloudy mixture was extracted with CH₂Cl₂ (4×5mL). The combined extracts were dried over anhydrous Na₂SO₄. Removal ofsolvent gave the desired product (90 mg, 95%) as white powder. m.p.111-112° C. IR (neat) 3316, 2937, 1667, 1650, 1556, 1454, 1348, 1102cm⁻¹; ¹H NMR (5% methanol-d4/CDCl₃, 300 MHz) δ 7.26-7.22 (m, 5 H), 4.37(bs, 3 H), 3.71-3.51(series of multiplets, 5 H), 3.44 (s, 2 H),3.39-3.10 (series of multiplets, 10 H), 2.27 (t, J=6.83 Hz, 2 H), 2.13(s, 3 H), 2.02-0.94 (series of multiplets, 33 H), 0.85 (d, J=5.62 Hz, 3H), 0.84 (s, 3 H), 0.61 (s, 3 H); ¹³C NMR (5% methanol-d4/CDCl₃, 75 MHz)δ 158.54, 158.48, 158.43, 138.27, 129.47, 128.32, 127.19, 81.89, 80.30,77.34, 69.02, 68.46, 67.21, 62.36, 58.00, 47.36, 46.18, is 43.26, 43.00,42.73, 42.18, 41.48, 39.32, 35.55, 34.97, 34.89, 34.67, 33.63, 28.93,28.28, 27.53, 27.16, 23.96, 23.28, 23.16, 22.77, 18.36, 12.58; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 753.5858 (100%), calcd.753.5867.

HCl salt of compound 4: Compound 4 was dissolved in minimum amount ofCH₂Cl₂ and MeOH followed by addition of excess HCl in ether. The solventwas removed by N₂ flow, and the residue was subjected to high vacuumovernight. The desired product was obtained as noncrystalline whitepowder. ¹H NMR (methanol-d4/20% CDCl₃, 300 MHz) δ 7.58 (bs, 2 H),7.50-7.48 (m, 3 H), 4.76 (bs, 13 H), 4.45 (d, J=12.9 Hz, 1 H), 4.27 (dd,1 H, J=12.9, 5.4 Hz), 3.82-3.00 (series of multiplets, 17 H), 2.81-2.80(m, 3 H), 2.20-1.02 (series of multiplets, 27 H), 0.98 (d, J=6.59 Hz, 3H), 0.95 (s, 3 H), 0.72 (s, 3 H); ¹³C NMR (methanol-d4/20% CDCl₃, 75MHz) δ 158.88, 158.72, 132.00, 131.96, 130.98, 130.15, 82.51, 81.07,78.05, 68.50, 68.02, 67.94, 67.10, 60.87, 60.53, 57.38, 47.16, 46.91,43.91, 43.11, 43.01, 42.91, 42.55, 40.28, 39.88, 39.95, 35.90, 35.73,35.64, 33.53, 29.18, 28.35, 27.99, 24.02, 23.30, 21.35, 18.52, 18.44,13.06.

Compound 5: A suspension of 2 (113 mg, 0.169 mmol) andaminoiminomethanesulfonic acid (67.1 mg, 0.541 mmol) in methanol andchloroform was stirred at room temperature for 24 hours. HCl in ether (1M, 1 mL) was added followed by the removal of solvent with N₂ flow. Theresidue was subject to high vacuum overnight and dissolved in H₂O (5 mL)followed by the addition of 20% NaOH solution (1.0 mL). The resultingmixture was extracted with CH₂Cl₂ (5×5 mL). The combined extracts weredried over anhydrous Na₂SO₄. Removal of solvent gave desired the product(90 mg, 95% yield) as a white solid. m.p. 102-104° C. IR (neat) 3332,3155, 2939, 2863, 1667, 1651, 1558, 1456, 1350, 1100 cm⁻¹; ¹H NMR (5%methanol-d4/CDCl₃, 300 MHz) δ 7.35-7.24 (m, 5 H), 3.75-3.64 (m, 1 H),3.57 (bs, 5 H), 3.50 (s, 2H), 3.53-3.46 (m, 1 H), 3.40-3.10 (series ofmultiplets, 14 H), 2.34 (t, J=7.31 Hz, 2 H), 2.19 (s, 3 H), 2.13-0.96(series of multiplets, 36H), 0.91 (bs, 6 H), 0.66 (s, 3 H); ¹³C NMR (5%methanol-d4/CDCl₃, 75 MHz) δ 157.49, 157.31, 157.23, 138.20, 129.52,128.34, 127.23, 81.17, 79.19, 76.42, 65.63, 65.03, 64.70, 62.36, 58.02,47.23, 46.24, 42.89, 42.18, 41.45, 39.45, 39.40, 39.30, 38.71, 35.61,35.55, 35.02, 34.82, 33.69, 29.87, 29.59, 29.42, 28.84, 27.96, 27.56,23.95, 23.40, 22.82, 22.64, 18.28, 12.54; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 795.6356 (84.3%), calcd. 795.6337.

HCl salt of compound 5: Compound 5 was dissolved in minimum amount ofCH₂Cl₂ and MeOH followed by the addition of excess HCl in ether. Thesolvent and excess HCl were removed by N₂ flow and the residue wassubject to high vacuum overnight. The desired product was obtained asnoncrystalline white powder. ¹H NMR (methanol-d4/10% CDCl₃, 300 MHz) δ7.62-7.54 (m, 2 H), 7.48-7.44 (m, 3 H), 4.84 (bs, 16 H), 4.46 (d, J=12.7Hz, 1 H), 4.26 (dd, J=12.7, 3.42 Hz, 1 H), 3.78-3.56 (series ofmultiplets, 5 H), 3.38-3.05 (series of multiplets, 13 H), 2.80 (d, 3 H),2.19-2.04 (m, 3 H), 2.02-1.04 (series of multiplets, 30 H), 0.98 (d,J=6.35 Hz, 3 H), 0.95 (s, 3 H), 0.72 (s, 3H); ¹³C NMR (methanol-d4/10%CDCl₃, 75 MHz) δ 158.75, 158.67, 132.32, 131.24, 130.83, 130.43, 82.49,81.02, 77.60, 66.47, 65.93, 61.19, 60.85, 57.69, 47.79, 47.60, 44.29,43.07, 40.86, 40.42, 40.19, 40.09, 39.76, 36.68, 36.50, 36.15, 35.94,33.91, 30.75, 30.46, 29.74, 29.33, 28.71, 24.41, 24.03, 23.38, 22.21,22.16, 18.59, 18.52, 13.09.

Example 2 Syntheses of Compounds 3, 28 and 29

Compound 28: A suspension of 19 (0.641 g, 0.614 mmol) and KCN (0.40 g,6.14 mmol) in anhydrous DMSO (5 mL) was stirred under N2 at 80° C.overnight followed by the addition of H₂O (50 mL). The aqueous mixturewas extracted with EtOAc (4×20 mL). The combined extracts were washedwith brine once, dried over anhydrous Na₂SO₄ and concentrated in vacuo.The residue was dissolved in CH₂Cl₂ (3 mL) and MeOH (3 mL) and catalyticamount of p-toluenesulfonic acid (5.84 mg, 0.03 mmol) was added. Thesolution was stirred at room temperature for 3 hours before theintroduction of saturated NaHCO₃ solution (10 mL). After the addition ofbrine (60 mL), the mixture was extracted with EtOAc (4×30 mL). Thecombined extracts were washed with brine once and dried over anhydrousNa₂SO₄ and concentrated. The residue afforded the desired product (0.342g, 92% yield) as pale yellowish oil after column chromatography (silicagel, EtOAc/hexanes 2:1). IR (neat) 3479, 2936, 2864, 2249, 1456, 1445,1366, 1348, 1108 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.76-3.53 (m, 7 H),3.32-3.06 (series of multiplets, 4 H), 2.57-2.46 (m, 6 H), 2.13-1.00(series of multiplets, 31 H), 0.93 (d, J=6.35 Hz, 3 H), 0.90 (s, 3 H),0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 119.91, 119.89, 80.75, 79.65,76.29, 65.83, 65.37, 65.19, 63.63, 46.57, 46.44, 42.77, 41.79, 39.71,35.63, 35.26, 35.02, 32.00, 29.46, 29.03, 27.96, 27.74, 26.64, 26.42,26.12, 23.56, 22.98, 22.95, 18.24, 14.65, 14.54, 14.30, 12.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 618.4247 (67.8%), calcd.618.4247.

Compound 29: To a solution of 28 (0.34 g, 0.57 mmol) in dry CH₂Cl₂ (15mL) under N₂ at 0° C. was added NEt₃ (119.5 μL, 0.857 mmol) followed bythe addition of mesyl chloride (53.1 μL, 0.686 mmol). The mixture wasallowed to stir at 0° C. for 30 minutes before the addition of H₂O (6mL). After the introduction of brine (60 mL), the aqueous mixture wasextracted with EtOAc (4×20 mL). The combined extracts were washed withbrine once, dried over anhydrous Na₂SO₄ and concentrated. To the residuewas added N-benzylmethyl amine (0.5 mL) and the mixture was stirredunder N₂ at 80° C. overnight. Excess N-benzylmethylamine was removed invacuo and the residue was subject to column chromatography (silica gel,EtOAc/hexanes 2:1 followed by EtOAc) to afford product (0.35 g, 88%yield) as a pale yellow oil. IR (neat) 2940, 2863, 2785, 2249, 1469,1453, 1366, 1348, 1108 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.21 (m,5H), 3.76-3.69 (m, 1 H), 3.64-3.50 (m, 4 H), 3.48 (s, 2 H), 3.31-3.05(series of multiplets, 4 H), 2.52-2.46 (m, 6 H), 2.33 (t, J=7.32 H, 2Hz), 2.18 (s, 3 H), 2.13-0.95 (series of multiplets, 30 H), 0.91 (d,J=6.80 H, 3 Hz), 0.90 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ139.37, 129.17, 128.26; 126.93, 119.96, 119.91, 80.73, 79.59, 76.26,65.79, 65.35, 65.13, 62.47, 58.25, 46.74, 46.40, 42.72, 42.38, 41.76,39.68, 35.78, 35.22, 34.98, 33.79, 28.99, 27.92, 27.71, 26.63, 26.38,26.09, 24.21, 23.54, 22.96, 22.90, 18.28, 14.62, 14.51, 14.26, 12.58;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 699.5226 (100%), calcd.699.5213.

Compound 3: A solution of 29 (0.074 g, 0.106 mmol) in anhydrous THF (10mL) was added dropwise to a mixture of AlCl₃ (0.1414 g, 1.06 mmol) andLiAlH₄ (0.041 g, 1.06 mmol) in dry THF (10 mL). The suspension wasstirred for 24 hours followed by the addition of 20% NaOH aqueoussolution (2 mL) at ice-bath temperature. Anhydrous Na₂SO₄ was added tothe aqueous slurry. The solution was filtered and the precipitate washedtwice with THF. After removal of solvent, the residue was subject tocolumn chromatography (silica gel, MeOH/CH₂Cl₂ 1:1 followed byMeOH/CH₂Cl₂/NH₃.H₂O 4:4:1) to afford the desired product (0.038 g, 50%yield) as a clear oil. IR (neat) 3362, 2935, 2863, 2782, 1651, 1574,1568, 1557, 1471, 1455, 1103 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.22(m, 5 H), 3.60-3.02 (series of broad multiplets, 18 H), 2.90-2.70 (m,5H), 2.33 (t, J=7.20 Hz, 2 H), 2.24-2.04 (m, 3 H), 2.18 (s, 3 H),1.96-0.96 (series of multiplets, 30 H), 0.90 (d, J=7.57 Hz, 3 H), 0.89(s, 3 H), 0.64 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 139.44, 129.24,128.31, 126.97, 80.63, 79.65, 75.97, 68.44, 68.00, 67.96, 62.54, 58.40,46.77, 46.30, 42.73, 42.43, 42.07, 41.92, 41.74, 41.72, 39.81, 35.82,35.48, 35.07, 33.84, 31.04, 30.30, 30.10, 29.03, 28.11, 27.82, 27.81,27.74, 27.67, 27.64, 24.31, 23.50, 23.04, 22.93, 18.22, 12.63; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 711.6139 (100%), calcd.711.6152; ([M+Na]⁺) 733.5974 (46.1%), calcd. 733.5972.

Example 3 Syntheses of Compounds 6, 7, and 30-33

Compound 30: Cholic acid (3.0 g, 7.3 mmol) was dissolved in CH₂Cl₂ (50mL) and methanol (5 mL). Dicyclohexylcarbodiimide (DCC) (1.8 g, 8.8mmol) was added followed by N-hydroxysuccinimide (˜100 mg) andbenzylmethylamine (1.1 g, 8.8 mmol). The mixture was stirred for 2hours, then filtered. The filtrate was concentrated and chromatographed(SiO₂, 3% MeOH in CH₂Cl₂) to give 3.0 g of a white solid (81% yield).m.p. 184-186° C.; IR (neat) 3325, 2984, 1678 cm⁻¹; ¹H NMR (CDCl₃, 200MHz) δ 7.21 (m, 5 H), 4.51 (m, 2 H), 3.87 (m, 1 H), 3.74 (m, 2 H), 3.36(m, 2 H), 2.84 (s, 3 H), 2.48-0.92 (series of multiplets, 28 H), 0.80(s, 3 H), 0.58 (d, J=6.5 Hz, 3 H); ¹³C NMR (CDCl₃, 50 MHz) δ 174.30,173.94, 137.36, 136.63, 128.81, 128.46, 127.85, 127.50, 127.18, 126.28,72.96, 71.76; 68.35, 53.39, 50.65, 48.77, 46.91, 46.33, 41.44, 39.36,39.18, 35.76, 35.27, 34.76, 33.87, 31.54, 34.19, 31.07, 30.45, 28.11,27.63, 26.14, 25.59, 24.92, 23.26, 17.51, 12.41; FAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 512 (100%), calcd. 512.

Compound 31: Compound 30 (2.4 g, 4.7 mmol) was added to a suspension ofLiAlH₄ (0.18 g, 4.7 mmol) in THF (50 mL). The mixture was refluxed for24 hours, then cooled to 0° C. An aqueous solution of Na₂SO₄ wascarefully added until the grey color of the mixture dissipated. Thesalts were filtered out, and the filtrate was concentrated in vacuo toyield 2.1 g of a white solid (88%). The product proved to be ofsufficient purity for further reactions. m.p. 70-73° C.; IR (neat) 3380,2983, 1502 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.23 (m, 5 H), 3.98 (bs, 2H), 3.81 (m, 3 H), 3.43 (m, 3 H), 2.74 (m, 2 H), 2.33 (m, 3 H), 2.25 (s,3 H), 2.10-0.90 (series of multiplets, 24 H), 0.98 (s, 3 H), 0.78 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 135.72, 129, 63, 128.21, 128.13, 125.28,72.91, 71.63, 62.05, 60.80, 56.79, 47.00, 46.23, 41.44, 40.81, 39.41,35.42, 35.24, 34.63, 34.02, 33.22, 31.73, 30.17, 29.33, 29.16, 28.02,27.49, 26.17, 25.55, 23.10, 22.48, 22.33, 17.54, 12.65; FAB-MS(thioglycerol matrix) m/e: ([M+H]⁺) 498 (100%), calcd. 498.

Compound 32: Compound 31 (0.36 g, 0.72 mmol) was dissolved in CH₂Cl₂ (15mL) and Bocglycine (0.51 g, 2.89 mmol), DCC (0.67 g, 3.24 mmol) anddimethylaminopyridine (DMAP) (˜100 mg) were added. The mixture wasstirred under N₂ for 4 hours then filtered. After concentration andchromatography (SiO₂, 5% MeOH in CH₂Cl₂), the product was obtained as a0.47 g of a clear glass (68%). ¹H NMR (CDCl₃, 300 MHz) δ 7.30 (m, 5 H),5.19 (bs, 1 H), 5.09 (bs, 3 H), 5.01 (bs, 1 H), 4.75 (m, 1 H), 4.06-3.89(m, 6 H), 2.33 (m, 2 H), 2.19 (s, 3 H) 2.05-1.01 (series of multiplets,26H), 1.47 (s, 9 H), 1.45 (s, 18 H), 0.92 (s, 3 H), 0.80 (d, J=6.4 Hz, 3H), 0.72 (s, 3 H). ¹³C NMR (CDCl₃, 75 MHz) δ 170.01, 169.86, 169.69,155.72, 155.55, 139.90, 129.05, 128.17, 126.88, 79.86, 76.53, 75.09,72.09, 62, 35, 57.88, 47.78, 45.23, 43.12, 42.79, 42.16, 40.81, 37.94,35.51, 34.69, 34.57, 34.36, 33.30, 31.31, 29.66, 28.80, 28.34, 27.22,26.76, 25.61, 24.02, 22.83, 22.47, 17.93, 12.19; FAB-MS (thioglycerolmatrix) m/e: ([M+H]⁺) 970 (100%), calcd. 970.

Compound 33: Compound 31 (0.39 g, 0.79 mmol) was dissolved in CH₂Cl₂ (15mL) and Boc-β-alanine (0.60 g, 3.17 mmol), DCC (0.73 g, 3.56 mmol) anddimethylaminopyridine (DMAP) (˜100 mg) were added. The mixture wasstirred under N₂ for 6 hours then filtered. After concentration andchromatography (SiO₂, 5% MeOH in CH₂Cl₂), the product was obtained as a0.58 g of a clear glass (72%). IR (neat) 3400, 2980, 1705, 1510 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 7.27 (m, 5 H), 5.12 (bs, 4 H), 4.93 (bs, 1 H),4.71 (m, 1 H), 3.40 (m, 12 H), 2.59-2.48 (m, 6 H), 2.28 (m, 2 H), 2.17(s, 3 H) 2.05-1.01 (series of multiplets, 26H), 1.40 (s, 27 H), 0.90 (s,3 H), 0.77 (d, J=6.1 Hz, 3H), 0.70 (s, 3 H). ¹³C NMR (CDCl₃, 75 MHz) δ171.85, 171.50, 171.44, 155.73, 138.62, 129.02, 128.09, 126.87, 79.18,75.53, 74.00, 70.91, 62.20, 57.67, 47.84, 44.99, 43.28, 41.98, 40.73,37.67, 36.12, 34.94, 34.65, 34.47, 34.20, 33.29, 31.23, 29.57, 28.74,28.31, 28.02, 27.86, 27.12, 26.73, 25.46, 24.86, 23.95, 22.77, 22.39,17.91, 12.14; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 1011.6619(100%), calcd. 1011.6634.

Compound 6: Compound 32 (0.15 g, 0.15 mmol) was stirred with excess 4 NHCl in dioxane for 40 minutes. The dioxane and HCl were removed in vacuoleaving 0.12 g of a clear glass (˜100%). ¹H NMR (CD₃OD, 300 MHz) δ 7.62(bs, 2 H), 7.48 (bs, 3 H), 5.30 (bs, 1 H), 5.11 (bs, 1 H), 4.72 (bs (1H), 4.46 (m, 1 H), 4.32 (m, 1 H) 4.05-3.91 (m, 4H), 3.10 (m, 2 H), 2.81(s, 3 H), 2.15-1.13 (series of multiplets, 25 H), 1.00 (s, 3 H), 0.91(bs, 3 H), 0.82 (s, 3 H). ¹³C NMR (CD₃OD, 125 MHz) δ 166.86, 166.50,131.09, 130.18, 129.17, 128.55, 76.60, 75.43, 72.61, 72.04, 70.40,66.22, 60.07, 58.00, 57.90, 54.89, 54.76, 46.44, 44.64, 43.39, 42.22,38.56, 36.78, 34.14, 33.92, 33.84, 31.82, 30.54, 29.67, 28.79, 27.96,26.79, 26.00, 24.99, 23.14, 22.05, 21.82, 19.91, 17.27, 11.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M−4 Cl−3 H]⁺) 669.4576 (100%), calcd.669.4591.

Compound 7: Compound 33 (0.20 g, 0.20 mmol) was stirred with excess 4 NHCl in dioxane for 40 minutes. The dioxane and HCl were removed in vacuoleaving 0.12 g of a clear glass (˜100%). ¹H NMR (CD₃OD, 500 MHz) δ 7.58(bs, 2 H), 7.49 (bs, 3 H), 5.21 (bs, 1 H), 5.02 (bs, 1 H), 4.64 (m, 1H), 4.44 (m, 1 H), 4.28 (m, 1 H), 3.30-2.84 (m, 14 H), 2.80 (s, 3 H),2.11-1.09 (series of multiplets, 25 H), 0.99 (s, 3 H), 0.89 (d, J=4.1Hz, 3 H), 0.80 (s, 3 H); ¹³C NMR (CD₃OD, 125 MHz) δ 171.92, 171.56,171.49, 132.44, 131.32, 131.02, 130.51, 78.13, 76.61, 61.45, 57.94,46.67, 44.80, 42.36, 40.85, 39.33, 37.03, 36.89, 36.12, 36.09, 35.79,35.63, 33.81, 33.10, 32.92, 32.43, 30.28, 28.43, 28.04, 26.65, 24.02,22.86, 21.98, 18.70, 12.68; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M−4 Cl−3 H]⁺) 711.5069 (43%), calcd. 711.5061.

Example 4 Syntheses of Compounds 8-10 and 34-40

Compound 34: Diisopropyl azodicarboxylate (DIAD) (1.20 mL, 6.08 mmol)was added to triphenylphosphine (1.60 g, 6.08 mmol) in THF (100 mL) at0° C. and was stirred for half an hour during which time the yellowsolution became a paste. Compound 14 (2.58 g, 4.06 mmol) andp-nitrobenzoic acid (0.81 g, 4.87 mmol) were dissolved in THF (50 mL)and added to the paste. The resulted mixture was stirred at ambienttemperature overnight. Water (100 mL) was added and the mixture was madeslightly basic by adding NaHCO₃ solution followed by extraction withEtOAc (3×50 mL). The combined extracts were washed with brine once anddried over anhydrous Na₂SO₄. The desired product (2.72 g, 85% yield) wasobtained as white powder after SiO₂ chromatography (Et₂O/hexanes 1:2).m.p. 207-209° C.; IR (KBr) 3434, 3056, 2940, 2868, 1722, 1608, 1529,1489, 1448, 1345 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 8.30-8.26 (m, 2 H),8.21-8.16 (m, 2 H), 7.46-7.42 (m, 6 H), 7.31-7.18 (m, 9 H)5.33 (bs, 1H), 4.02 (bs, 1 H), 3.90 (bs, 1 H), 3.09-2.97 (m, 2 H), 2.68 (td,J=14.95, 2.56 Hz, 1 H), 2.29-2.19 (m, 1 H), 2.07-1.06 (series ofmultiplets, 24 H), 1.01 (s, 3 H), 0.98 (d, J=6.6 Hz, 3 H), 0.70 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 164.21, 150.56, 144.70, 136.79, 130.77,128.88, 127.86, 126.98, 123.70, 86.47, 73.24, 73.00, 68.70, 64.22,47.79, 46.79, 42.15, 39.76, 37.47, 35.52, 35.34, 34.23, 33.79, 32.46,31.12, 28.74, 27.71, 26.85, 26.30, 25.16, 23.41, 17.98, 12.77; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 808.4203 (53.8%), calcd.808.4189. The nitrobenzoate (2.75 g, 3.5 mmol) was dissolved in CH₂Cl₂(40 mL) and MeOH (20 mL) and 20% aqueous NaOH (5 mL) were added. Themixture was heated up to 60° C. for 24 hours. Water (100 mL) wasintroduced and extracted with EtOAc. The combined extracts were washedwith brine and dried over anhydrous Na₂SO₄. The desired product (1.89 g,85% yield) was obtained as white solid after SiO₂ chromatography (3%MeOH in CH₂Cl₂ as eluent). m.p. 105-106° C.; IR (KBr) 3429, 3057, 2936,1596, 1489, 1447, 1376, 1265, 1034, 704 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ7.46-7.42 (m, 6 H), 7.32-7.19 (m, 9 H), 4.06 (bs, 1 H), 3.99-(bs, 1 H),3.86 (bd, J=2.44 Hz, 1 H), 3.09-2.97 (m, 2 H), 2.47 (td, J-=14.03, 2.44Hz, 1 H), 2.20-2.11 (m, 1H), 2.04-1.04 (series of multiplets, 25 H),0.97 (d, J=6.59 Hz, 3 H), 0.94 (s, 3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃,75 MHz) δ 144.70, 128.88, 127.86, 126.97, 86.45, 73.31, 68.84, 67.10,64.23, 47.71, 46.74, 42.10, 39.70, 36.73, 36.73, 36.15, 35.53, 35.45,34.45, 32.46, 29.93, 28.67, 27.86, 27.71, 26.87, 26.04, 23.43, 23.16,17.94, 12.75; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 659.4064(100%), calcd. 659.4076.

Compound 35: To a round-bottom flask were added 34 (2.0 g, 3.14 mmol),NaH (60% in mineral oil, 3.8 g, 31.4 mmol) and THF (150 mL). Thesuspension was refluxed for 2 hours followed by the addition of allylbromide (2.72 mL, 31.4 mL). After refluxing for 28 hours, another 10 eq.of NaH and allyl bromide were added. After 72 hours, another 10 eq. ofNaH and allyl bromide were added. After 115 hours, TLC showed almost nostarting material or intermediates. Water (100 mL) was added to thesuspension carefully, followed by extraction with EtOAc (5×50 mL). Thecombined extracts were washed with brine and dried over anhydrousNa₂SO₄. The desired product (1.81 g, 79% yield) was obtained as ayellowish glass after SiO₂ chromatography (5% EtOAc/hexanes). IR (neat)3060, 3020, 2938, 2865, 1645, 1596, 1490, 1448, 1376, 1076, 705 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6 H), 7.31-7.18 (m, 9 H), 6.06-5.85(m, 3 H), 5.35-5.20 (m, 3 H), 5.15-5.06 (m, 3 H), 4.10-4.00 (m, 2 H),3.93-3.90 (m, 2 H), 3.85-3.79 (ddt, J=13.01, 4.88, 1.59 Hz, 1 H),3.73-3.66 (ddt, i=13.01, 5.38, 1.46 Hz, 1 H), 3.58 (bs, 1 H), 3.54 (bs,1 H), 3.32 (d, J=2.93 Hz, 1 H), 3.07-2.96 (m, 2 H), 2.36 (td, J=13.67,2.68 Hz, 1 H), 2.24-2.10 (m, 2 H), 2.03-1.94 (m, 1 H), 1.87-0.86 (seriesof multiplets, 20 H), 0.91 (s, 3 H), 0.90 (d, J=6.83 Hz, 3 H), 0.64 (s,3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.77, 136.29, 136.21, 136.13, 128.90,127.86, 126.94, 116.13, 115.51, 115.42, 86.44, 81.11, 75.65, 73.92,69.40, 68.81, 64.43, 46.68, 46.54, 42.93, 39.93, 36.98, 35.66, 35.14,35.14, 32.83, 32.54, 30.48, 28.51, 27.72, 27.64, 26.82, 24.79, 23.65,23.43, 23.40, 18.07, 12.80; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+H]⁺) 757.5185 (12.9%), calcd. 757.5196.

Compound 36: Ozone was bubbled through a solution of 35 (0.551 g, 0.729mmol) in CH₂Cl₂ (40 mL) and MeOH (20 mL) at −78° C. until the solutionturned a deep blue. Excess ozone was blown off with oxygen.Methylsulfide (1 mL) was added followed by the addition of NaBH₄ (0.22g, 5.80 mmol) in 5% NaOH solution and methanol. The resulted mixture wasstirred overnight at room temperature and washed with brine. The brinewas then extracted with EtOAc (3×20 mL). The combined extracts weredried over Na₂SO₄. The desired product (0.36 g, 65% yield) was obtainedas a colorless glass after SiO₂ chromatography (5% MeOH/CH₂Cl₂). IR(neat) 3396, 3056, 2927, 1596, 1492, 1462, 1448, 1379, 1347, 1264, 1071cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6 H), 7.32-7.18 (m, 9 H),3.77-3.57 (series of multiplets, 10 H) 3.48-3.44 (m, 2 H), 3.36-3.30 (m,2 H), 3.26-3.20 (m, 1 H), 3.04-2.99 (m, 2 H), 2.37-0.95 (series ofmultiplets, 27 H), 0.92 (s, 3 H), 0.91 (d, J=6.59 Hz, 3 H), 0.67 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.69, 128.87, 127.84, 126.94, 86.44,81.05, 76.86, 74.65, 69.91, 69.22, 68.77, 64.24, 62.44, 62.42, 62.26,46.92, 46.54, 42.87, 39.73, 36.86, 35.52, 35.13, 32.82, 32.54, 30.36,28.71, 27.61, 27.44, 26.79, 24.82, 23.51, 23.38, 23.31, 18.28, 12.74;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 791.4844 (96.4%),calcd. 791.4863.

Compound 37: NEt₃ (0.23 mL, 1.66 mmol) was added to a solution of 36(0.364 g, 0.47 mmol) in dry CH₂Cl₂ (30 mL) at 0° C. under N₂ followed bythe introduction of mesyl chloride (0.12 mL, 1.56 mmol). The mixture wasstirred for 10 minutes and H₂O (10 mL) added to quench the reaction,followed by extraction with EtOAc (3×30 mL). The combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. SiO₂ chromatography(EtOAc/hexanes 1:1) gave the desired product (0.41 g, 86% yield) aswhite glass. IR (neat) 3058, 3029, 2939, 2868, 1491, 1461, 1448, 1349,1175, 1109, 1019 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6 H),7.31-7.19 (m, 9 H), 4.35-4.26 (m, 6 H), 3.84-3.74 (m, 2 H), 3.64-3.56(m, 4 H), 3.49-3.34 (m, 3 H), 3.06 (s, 3 H), 3.04 (s, 3 H), 3.02 (s, 3H), 3.09-2.95 (m, 2 H), 2.28 (bt, J=14.89 Hz, 1 H), 2.09-0.86 (series ofmultiplets, 21 H), 0.92 (s, 3 H), 0.90 (d, J=6.78 Hz, 3 H), 0.66 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.66, 128.86, 127.86, 126.97, 86.46,81.28, 77.18, 75.00, 70.14, 69.89, 69.13, 66.49, 65.85, 65.72, 64.22,47.06, 46.35, 42.77, 39.58, 37.81, 37.64, 37.55, 36.75, 35.48, 35.02,32.59, 32.52, 30.27, 28.43, 27.56, 27.52, 26.92, 24.62, 23.34, 23.25,23.10, 18.24, 12.64; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)1025.4207 (100%), calcd. 1025.4189.

Compound 38: The suspension of 37 (0.227 g, 0.227 mmol) and NaN₃ (0.147g, 2.27 mmol) in dry DMSO (5 mL) was stirred at 80° C. overnight,diluted with H₂O (50 mL) and extracted with EtOAc (3×20 mL). Theextracts were washed with brine once and dried over anhydrous Na₂SO₄.SiO₂ chromatography (EtOAc/hexanes 1:8) afforded the desired product(0.153 g, 80% yield) as a yellow oil. IR (neat) 2929, 2866, 2105, 1490,1466, 1448, 1107, 705 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.42 (m, 6H), 7.32-7.19 (m, 9 H), 3.80-3.74 (m, 1 H), 3.70-3.55 (series ofmultiplets, 5 H), 3.41-3.19 (series of multiplets, 9 H), 3.04-2.98 (m, 2H), 2.41 (td, J=13.1, 2.44 Hz, 1 H), 2.29-2.14 (m, 2 H), 2.04-0.86(series of multiplets, 20 H), 0.93 (s, 3 H), 0.91 (d, J=6.60 Hz, 3 H),0.66 (s, 3. H); ¹³C NMR (CDCl₃, 75 MHz) δ 144.78, 128.93, 127.87,126.96, 86.46, 81.30, 77.16, 75.21, 67.99, 67.44, 67.03, 64.41, 51.64,51.57, 51, 33, 46.71, 46.30, 42.35, 39.75, 36.72, 35.64, 35.20, 32.52,32.42, 30.17, 28.63, 27.80, 27.22, 26.90, 24.80, 23.55, 23.30, 23.24,18.23, 12.65; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 866.5049(96.9%), calcd. 866.5057.

Compound 39: p-Toluenesulfonic acid (1.72 mg) was added into thesolution of 38 (0.153 g, 0.18 mmol) in CH₂Cl₂ (5 mL) and MeOH (5 mL),and the mixture was stirred for 2.5 hours. Saturated NaHCO₃ solution (5mL) was introduced followed by the introduction of brine (30 mL). Theaqueous mixture was extracted with EtOAc and the combined extractswashed with brine and dried over Na₂SO₄. The desired product (0.10 g,92% yield) was obtained as a pale yellowish oil after SiO₂chromatography (EtOAc/hexanes 1:3). IR (neat) 3426, 2926, 2104, 1467,1441, 1347, 1107 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.81-3.74 (m, 1 H),3.71-3.54 (m, 7 H), 3.41-3.19 (m, 9 H), 2.41 (td, J=13.61, 2.32 Hz, 1H), 2.30-2.14 (m, 2 H), 2.07-1.98 (m, 1 H), 1.94-0.95 (series ofmultiplets, 21 H), 0.95 (d, J=6.35 Hz, 3 H), 0.93 (s, 3 H), 0.69 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 81.22, 77.08, 75.13, 67.94, 67.36, 66.97,63.76, 51.59, 51.51, 51.26, 46.51, 46.24, 42.31, 39.68, 36.64, 35.58,35.12, 32.34, 31.92, 30.11, 29.55, 28.54, 27.82, 27.16, 24.75, 23, 47,23.23, 23.18, 18.15, 12.56; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 624.3966 (54.9%), calcd. 624.3962.

Compound 40: To a solution of 39 (0.10 g, 0.166 mmol) in CH₂Cl₂ (8 mL)at 0° C. was added NEt₃ (34.8 μL, 0.25 mmol) under N₂ followed by theintroduction of mesyl chloride (15.5 μL, 0.199 mmol). The mixture wasstirred 15 minutes. Addition of H₂O (3 mL) and brine (20 mL) wasfollowed by extraction with EtOAc (4×10 mL). The combined extracts werewashed with brine once and dried over Na₂SO₄. After removal of solvent,the residue was mixed with N-benzylmethylamine (0.5 mL) and heated to80° C. under N₂ overnight. Excess N-benzyl methylamine was removed invacuo and the residue was subjected to SiO₂ chromatography(EtOAc/hexanes 1:4) to give the product (0.109 g, 93% yield) as a yellowoil. IR (neat) 2936, 2784, 2103, 1467, 1442, 1346, 1302, 1106, 1027cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 7.32-7.23 (m, 5 H), 3.81-3.74 (m, 1 H),3.71-3.55 (m, 5 H), 3.47 (s, 2 H), 3.41-3.19 (m, 9 H), 2.46-2.11 (m, 5H), 2.18 (s, 3H), 2.03-0.85 (series of multiplets, 20 H), 0.93 (s, 3 H),0.93 (d, J=6.35 Hz, 3 H,), 0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ139.54, 129.26, 128.32, 126.97, 81.26, 77.12, 75.17, 67.98, 67.42,67.00, 62.50, 58.41, 51.61, 51.54, 51.29, 46.66, 46.28, 42.46, 42.32,39.72, 36.68, 35.76, 35.16, 33.75, 32.38, 30.15, 28.59, 27.85, 27.19,24.77, 24.15, 23.53, 23.28, 23.22, 18.28, 12.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 705.4929 (100%), calcd.705.4928.

Compound 8: A suspension of 40 (0.109 g, 0.155 mmol) and LiAlH₄ (23.5mg, 0.62 mmol) in THF (20 mL) was stirred under N₂ overnight. Na₂SO₄ 10H₂O was carefully added and stirred until no grey color persisted.Anhydrous Na₂SO₄ was added and the white precipitate was filtered outand rinsed with dry THF. After removal of solvent, the residue wasdissolved in minimum CH₂Cl₂ and filtered. The desired product (0.091 g,94% yield) was obtained as a colorless oil after the solvent wasremoved. IR (neat) 3371, 3290, 3027, 2938, 2862, 2785, 1586, 1493, 1453,1377, 1347, 1098 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz)_b 7.31-7.21 (m, 5 H),3.65-3.53 (m, 4 H), 3.47 (s, 2 H), 3.42-3.34 (m, 2 H), 3.30 (bs, 1 H),3.26-3.20 (m, 1 H), 3.14-3.09 (m, 1 H), 2.89-2.81 (m, 6 H), 2.39-2.27(m, 3 H), 2.17 (s, 3 H), 2.15-0.88 (series of multiplets, 29 H), 0.93(d, J=6.59 Hz, 3 H), 0.92 (s, 3 H), 0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75MHz) δ 139.34, 129.16, 128.24, 126.90, 80.75, 76.44, 74.29, 70.58,69.88, 69.75, 62.47, 58.27, 46.66, 46.47, 42.75, 42.63, 42.51, 42.35,39.77, 36.87, 35.73, 35.04, 33.77, 32.90, 30.38, 28.71, 27.70, 27.32,24.89, 24.09, 23.53, 23.36, 23.25, 18.24, 12.62; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 627.5199 (23.3%), calcd.627.5213.

Compound 9: To a solution of 23 (0.18 g, 0.28 mmol) in dry DMF (4 mL)were added NaH (0.224 g, 60% in mineral oil, 5.60 mmol) and 1-bromooctane (0.48 mL, 2.80 mmol). The suspension was stirred under N₂ at 65°C. overnight followed by the introduction of H₂O (60 mL) and extractionwith ether (4×20 mL). The combined extracts were washed with brine anddried over Na₂SO₄. SiO₂ chromatography (hexanes and 5% EtOAc in hexanes)afforded the desired product (0.169 g, 80% yield) as a pale yellowishoil. IR (neat) 2927, 2865, 2099, 1478, 1462, 1451, 1350, 1264, 1105cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.69-3.35 (series of multiplets, 15 H),3.26-3.02 (series of multiplets, 4 H), 2.19-2.02 (m, 3 H), 1.97-1.16(series of multiplets, 37 H), 1.12-0.99 (m, 2 H), 0.92-0.86 (m, 9 H),0.65 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.69, 79.84, 76.13, 71.57,71.15, 65.07, 64.49, 64.39, 49.08, 48.99, 48.80, 46.68, 46.45, 42.72,42.05, 39.88, 35.74, 35.49, 35.36, 35.14, 32.42, 32.03, 30.01, 29.85,29.81, 29.76, 29.67, 29.48, 29.14, 27.92, 27.80, 27.70, 26.58, 26.42,23.59, 23.09, 22.92, 22.86, 18.11, 14.31, 12.65, HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 778.5685 (22.1%), calcd.778.5683. The triazide (0.169 g, 0.224 mmol) and LiAlH₄ (0.025 g, 0.67mmol) were suspended in anhydrous THF (10 mL) and stirred under N₂ atroom temperature overnight followed by careful introduction of Na₂SO₄hydrate. After the grey color disappeared, anhydrous Na₂SO₄ was addedand stirred. The white precipitate was removed by filtration and washedwith TBF. After removal of solvent, the residue was dissolved in 1 Mhydrochloric acid and the aqueous solution was extracted with ether (5mL) once. The aqueous solution was then made basic by adding 20% aqueousNaOH solution followed by extraction with Et₂O (4×5 mL). The combinedextracts were washed, dried and concentrated. The residue was thensubject to SiO₂ chromatography (MeOH/CH₂Cl₂ (1:1) followed byMeOH/CH₂Cl₂/NH₃.H₂O (4:4:1)) to afford the desired product (0.091 g, 60%yield) as a colorless oil. IR (neat) 3361, 2927, 2855, 1576, 1465, 1351,1105 cm⁻¹; ¹H NMR (CD₃OD, 300 MHz) δ 4.86 (bs, 6 H), 3.77-3.72 (m, 1 H),3.70-3.61 (m, 1 H), 3.57-3.53 (m, 3 H), 3.43-3.38 (m, 4 H), 3.34-3.27(m, 2 H), 3.18-3.10 (m, 2 H), 2.84-2.71 (m, 6 H), 2.22-2.07 (m, 3 H),2.00-1.02 (series of multiplets, 39 H), 0.97-0.88 (m, 9 H), 0.71 (s, 3H); ¹³C NMR (CD₃OD, 75 MHz) δ 82.20, 81.00, 77.62, 72.52, 72.06, 68.00,67.92, 67.39, 48.20, 47.53, 44.26, 43.40, 41.42, 41.15, 40.84, 40.35,36.88, 36.73, 36.42, 36.11, 34.24, 34.05, 33.94, 33.67, 33.17, 30.95,30.72, 30.62, 29.81, 29.35, 28.87, 28.79, 27.51, 24.57, 23.90, 23.83,23.44, 18.76, 14.62, 13.07; HRFAB-MS (thioglycerol matrix) m/e: ([M+H]⁺)678.6133 (100%), calcd. 678.6149.

Compound 10: A suspension of 23 (0.126 g, 0.196 mmol) and LiAlH₄ (0.037g, 0.98 mmol) in THF (40 mL) was stirred at room temperature under N₂overnight followed by careful addition of Na₂SO₄.10 H₂O. After the greycolor in the suspension disappeared, anhydrous Na₂ SO₄ was added andstirred until organic layer became clear. The white precipitate wasremoved by filtration and washed with twice THF. The THF was removed invacuo, and the residue was subject to SiO₂ chromatography(MeOH/CH₂Cl₂/NH₃.H₂O (4:4:1)) to afford the desired product (0.066 g,60% yield) as a colorless oil. IR (neat) 3365, 2933, 2865, 1651, 1471,1455, 1339, 1103 cm⁻¹; ¹H NMR (CDCl₃/30% CD₃OD, 300 MHz) δ 4.43 (bs, 7H), 3.74-3.68 (m, 1 H), 3.66-3.60 (m, 1H), 3.57-3.50 (m, 5 H), 3.34-3.25(M, 2 H), 3.17-3.06 (M, 2 H), 2.84-2.74 (M, 6 H), 2.19-2.01 (M, 3 H),1.97-0.96 (series of multiplets, 27 H), 0.94 (d, J=7.2 Hz, 3 H), 0.92(s, 3 H), 0.69 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.44, 79.27, 75.77,66.59, 66.53, 65.86, 62.51, 46.21, 45.84, 42.55, 41.53, 40.09, 39.43,39.31, 39.02, 35.16, 34.93, 34.86, 34.57, 32.93, 32.71, 31.57, 28.66,28.33, 27.64, 27.22, 23.04, 22.40, 22.29, 17.60, 11.98; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 566.4889 (8.9%), calcd.566.4897.

Example 5 Syntheses of Compounds 11 and 43-47

Compound 43: Compound 41 was prepared following the method reported byD. H. R. Barton, J. Wozniak, S. Z. Zard, A SHORT AND EFFICIENTDEGRADATION OF THE BILE ACID SIDE CHAIN. SOME NOVEL REACTIONS OFSULPHINES AND A-KETOESTERS, Tetrahedron, 1989, vol. 45, 3741-3754. Amixture of 41 (1.00 g, 2.10 mmol), ethylene glycol (3.52 mL, 63 mmol)and p-TsOH (20 mg, 0.105 mmol) was refluxed in benzene under N₂ for 16hours. Water formed during the reaction was removed by a Dean-Starkmoisture trap. The cooled mixture was washed with NaHCO₃ solution (50mL) and extracted with Et₂O (50 mL, 2×30 mL). The combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. Removal of thesolvent gave the product (1.09 g, 100%) as a white glass. IR (neat)2939, 2876, 1735, 1447, 1377, 1247, 1074, 1057, 1039 cm⁻¹; ¹H NMR(CDCl₃, 300 MHz) δ 5.10 (t, J=2.70 Hz, 1 H), 4.92 (d, J=2.69 Hz, 1 H),4.634.52 (m, 1 H), 3.98-3.80 (m, 4 H), 2.32 (t, J=9.51 Hz, 1H), 2.13 (s,3 H), 2.08 (s, 3 H); 2.05 (s, 3 H), 2.00-1.40 (series of multiplets, 15H), 1.34-0.98(m, 3 H), 1.20(s, 3 H), 0.92(s, 3 H), 0.82(s, 3 H); ¹³CNMR(CDCl₃, 75 MHz) 170.69, 170.63, 170.47, 111.38, 75.07, 74.23, 70.85,64.95, 63.43, 49.85, 44.73, 43.39, 41.11, 37.37, 34.84, 34.80, 34.52,31.42, 29.18, 27.02, 25.41, 24.16, 22.72, 22.57, 22.44, 21.73, 21.63,13.40; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 521.3106(38.6%), calcd. 521.3114. The triacetate (1.09 g, 2.10 mmol) wasdissolved in MeOH (50 mL). NaOH (0.84 g, 21 mmol) was added to thesolution. The suspension was then refluxed under N₂ for 24 hours. MeOHwas then removed in vacuo and the residue was dissolved in Et₂O (100 mL)and washed with H₂O, brine, and then dried over anhydrous Na₂SO₄. Thedesired product (0.80 g, 96% yield) was obtained as white solid afterremoval of solvent. m.p. 199-200° C. IR (neat) 3396, 2932, 1462, 1446,1371, 1265, 1078, 1055 cm⁻¹; ¹H NMR (10% CD₃OD in CDCl₃, 300 MHz) δ4.08-3.83 (series of multiplets, 9 H), 3.44-3.34 (m, 1 H), 2.41 (t,J=9.28 Hz, 1 H), 2.22-2.10 (m, 2 H), 1.96-1.50 (series of multiplets, 12H), 1.45-0.96 (series of multiplets, 4 H), 1.32 (s, 3 H) 0.89 (s, 3 H),0.78 (s, 3 H); ¹³C NMR (10% CD₃OD in CDCl₃, 75 MHz) δ 112.11, 72.35,71.57, 68.09, 64.54, 63.24, 49.36, 45.90, 41.48, 41.45, 39.18, 38.79,35.29, 34.71, 34.45, 29.90, 27.26, 26.60, 23.65, 22.54, 22.44, 22.35,13.46; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 417.2622(87.3%), calcd. 417.2617.

Compound 44: To a round-bottom flask were added 43 (0.80 g, 2.03 mmol)and dry THF (100 mL) followed by the addition of NaH (60% in mineraloil, 0.81 g, 20.3 mmol). The suspension was refluxed under N₂ for 30minutes before the addition of allyl bromide (1.75 mL, 20.3 mmol). After48-hours of reflux, another 10 eq. of NaH and allyl bromide were added.After another 48 hours, TLC showed no intermediates left. Cold water (50mL) was added to the cooled suspension. The resulted mixture wasextracted with Et₂O (60 mL, 2×30 mL). The combined extracts were washedwith brine and dried over anhydrous Na₂SO₄. SiO₂ column chromatography(6% EtOAc in hexanes) gave the desired product (0.94 g, 90% yield) as apale yellow oil. IR (neat) 3076, 2933, 2866, 1645, 1446, 1423, 1408,1368, 1289, 1252, 1226, 1206, 1130, 1080, 1057 cm⁻¹; ¹H NMR (CDCl₃, 300MHz) δ 6.02-5.84 (m, 3 H), 5.31-5.04 (m, 6 H), 4.12-4.05 (m, 2 H),4.01-3.81 (m, 7 H), 3.70 (dd, J=12.94, 5.62 Hz, 1 H), 3.55 (t, J=2.56Hz, 1 H), 3.33 (d, J-=2.93 Hz, 1 H), 3.18-3.08 (m, 1 H), 2.65 (t,J=10.01 Hz, 1 H), 2.32-2.14 (m, 3 H), 1.84-1.45 (series of multiplets,10 H), 1.41-1.22 (m, 3 H), 1.27 (s, 3 H), 1.14-0.92 (m, 2 H), 0.89 (s, 3H), 0.75 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 136.38, 136.07, 136.00,116.31, 115.54, 115.38, 112.34, 80.07, 79.22, 75.05, 69.83, 69.34,68.82, 65.14, 63.24, 48.80, 45.96, 42.47, 42.15, 39.40, 35.55, 35.16,35.15, 29.04, 28.22, 27.52, 24.21, 23.38, 23.11, 22.95, 22.58, 13.79;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 537.3549 (100%),calcd. 537.3556.

Compound 45: To the solution of 44 (0.94 g, 1.83 mmol) in dry THF (50mL) was added 9-BBN (0.5 M solution in THF, 14.7 mL, 7.34 mmol) and themixture was stirred under N₂ at room temperature for 12 hours before theaddition of 20% NaOH solution (4 mL) and 30% H₂O₂ solution (4 mL). Theresulted mixture was then refluxed for an hour followed by the additionof brine (100 mL) and extracted with EtOAc (4×30 mL). The combinedextracts were dried over anhydrous Na₂SO₄. After the removal of solvent,the residue was purified by SiO₂ column chromatography (EtOAc followedby 10% MeOH in CH₂Cl₂) to give the product (0.559 g, 54% yield) as acolorless oil. IR (neat) 3410, 2933, 2872, 1471, 1446, 1367, 1252, 1086cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 4.02-3.52 (series of multiplets, 17 H),3.41-3.35 (m, 1 H), 3.29 (d, J=2.44 Hz, 1H), 3.22-3.15 (m, 3 H), 2.58(t, J=10.01 Hz, 1 H), 2.27-1.95 (m, 3 H), 1.83-1.48 (series ofmultiplets, 16 H), 1.40-0.93 (series of multiplets, 5 H), 1.27 (s, 3 H),0.90 (s, 3 H), 0.75 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 112.41, 80.09,79.09, 76.31, 66.70, 66.02, 65.93, 64.80, 63.26, 61.53, 61.25, 60.86,48.59, 45.80, 42.51, 41.72, 39.10, 35.36, 35.02, 34.98, 32.87, 32.52,32.40, 28.88, 27.94, 27.21, 24.33, 23.02, 22.84 (2 C's), 22.44, 13.69;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 591.3881 (100%),calcd. 591.3873.

Compound 46: To a solution of 45 (0.559 g, 0.98 mmol) in acetone (40 mL)and water (4 mL) was added PPTS (0.124 g, 0.49 mmol) and the solutionwas refluxed under N₂ for 16 hours. The solvent was removed underreduced pressure. Water (40 mL) was then added to the residue and themixture was extracted with EtOAc (40 mL, 2×20 mL). The combined extractswere washed with brine, dried and evaporated to dryness. SiO₂ columnchromatography (8% MeOH in CH₂Cl₂) of the residue afforded the desiredproduct (0.509 g, 98% yield) as clear oil. IR (neat) 3382, 2941, 2876,1699, 1449, 1366, 1099 cm⁻¹; ¹H NMR (CDCl₃, 300 MHz) δ 3.83-3.72 (m, 8H), 3.66 (t, J=5.62 Hz, 2 H), 3.54 (bs, 2 H), 3.43-3.28 (m, 4 H),3.24-3.12 (m, 2 H), 2.26-2.00 (m, 4 H), 2;08 (s, 3 H), 1.98-1.50 (seriesof multiplets, 15 H), 1.42-0.96 (series of multiplets, 6 H), 0.90 (s, 3H), 0.62 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 210.49, 78.87 (2 C's),76.30, 66.86, 66.18, 65.69, 61.74, 61.43, 60.71, 55.31, 48.05, 43.02,41.58, 39.53, 35.28, 35.09, 34.96, 32.77, 32.70, 32.31, 31.12, 28.72,27.88, 27.14, 23.47, 22.75, 22.47, 22.34, 13.86; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 547.3624 (100%), calcd.547.3611.

Compound 47: To a solution of 46 (0.18 g, 0.344 mmol) in dry CH₂Cl₂ (10mL) at 0° C. was added Et₃N (0.168 mL, 1.20 mmol) followed by theaddition of mesyl chloride (0.088 mL, 1.13 mmol). After 10 minutes, H₂O(3 mL) and brine (30 mL) were added. The mixture was extracted withEtOAc (30 mL, 2×10 mL) and the extracts were washed with brine and driedover anhydrous Na₂SO₄. After removal of solvent, the residue wasdissolved in DMSO (5 mL) and NaN₃ (0.233 g, 3.44 mmol). The suspensionwas heated up to 50° C. under N₂ for 12 hours. H₂O (50 mL) was added tothe cool suspension and the mixture was extracted with EtOAc (30 mL,2×10 mL) and the extracts were washed with brine and dried overanhydrous Na₂SO₄. SiO₂ column chromatography (EtOAc/hexanes 1:5)afforded the product (0.191 g, 88% yield for two steps) as a pale yellowoil. IR (neat) 2933, 2872, 2096, 1702, 1451, 1363, 1263, 1102 cm⁻¹; ¹HNMR (CDCl₃, 300 MHz). δ 3.72-3.64 (m, 2 H), 3.55-3.24 (series ofmultiplets, 11 H), 3.18-3.02 (m, 2 H), 2.22-2.02 (m, 4 H), 2.08 (s, 3H), 1.95-1.46 (series of multiplets, 15 H), 1.38-0.96 (series ofmultiplets, 6 H), 0.89 (s, 3 H), 0.62 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz)δ 210.36, 79.69, 79.22, 75.98, 65.08, 64.80, 64.53, 55.31, 48.93, 48.86,48.76, 48.06, 43.03, 41.91, 39.66, 35.44, 35.31, 35.12, 31.04, 29.77,29.69, 29.67, 28.99, 28.10, 27.65, 23.60, 22.99, 22.95, 22.50, 14.00;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 622.3820 (100%),calcd. 622.3805.

Compound 11: Compound 47 (0.191 g, 0.319 mmol) was dissolved in dry ThF(20 mL) followed by the addition of LiAlH₄ (60.4 mg, 1.59 mmol). Thegrey suspension was stirred under N₂ at room temperature for 12 hours.Na₂SO₄.10H₂O powder was carefully added. After the grey color in thesuspension disappeared, anhydrous Na₂SO₄ was added and the precipitatewas filtered out. After the removal of solvent, the residue was purifiedby column chromatography (silica gel, MeOH/CH₂Cl₂/28% NH₃—H₂O 3:3:1).After most of the solvent was rotavapped off from the fractionscollected, 5% HCl solution (2 mL) was added to dissolve the milkyresidue. The resulted clear solution was then extracted with Et₂O (2×10mL). 20% NaOH solution was then added until the solution became stronglybasic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄ and removal ofsolvent gave the desired product (0.115 g, 69% yield) as a colorlessoil. From ¹H NMR it appears that this compound was a mixture of twostereoisomers at C₂₀ with a ratio of approximately 9:1. Thestereoisomers were not separated, but used as recovered. Spectra for themost abundant isomer: IR (neat) 3353, 2926, 2858, 1574, 1470, 1366, 1102cm⁻¹; ¹H NMR (20% CDCl₃ in CD₃OD, 300 MHz) δ 4.69 (bs, 7 H), 3.76-3.69(m, 1 H), 3.63-3.53 (m, 5 H), 3.50-3.40 (m, 1 H), 3.29 (bs, 1 H),3.18-3.07 (m, 2 H), 2.94-2.83 (m, 1 H), 2.81-2.66 (m, 5 H), 2.23-2.06(m, 4 H), 1.87-1.50 (series of multiplets, 15 H), 1.39-0.96 (series ofmultiplets, 6 H), 1.11 (d, J=6.10 Hz, 3 H), 0.93 (s, 3 H), 0.75 (s, 3H); ¹³C NMR (20% CDCl₃ in CD₃OD, 75 MHz) δ 81.46, 80.67, 77.32, 70.68,67.90, 67.66, 67.18, 50.32, 47.17, 43.30, 43.06, 40.74, 40.64, 40.38,40.26, 36.31, 36.28, 35.93, 34.30, 34.02, 33.29, 29.63, 29.31, 28.43,26.10, 24.67, 24.09, 23.96, 23.50, 13.30 for the major isomer; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 524.4431 (64.2%), calcd.524.4427.

Example 6 Syntheses of Compounds 12, 48 and 49

Compound 48: To a solution of 23 (0.15 g, 0.233 mmol) in dq CH₂Cl₂ (15mL) at 0° C. was added Et₃N (48.8 μL, 0.35 mmol) followed by theaddition of CH₃SO₂Cl (21.7 μL, 0.28 mmol). The mixture was stirred for15 minutes before H₂O (3 mL) was added. Saturated NaCl solution (20 mL)was then added, and the mixture was extracted with EtOAc (40 mL, 2×20mL). The combined extracts were washed with brine and dried overanhydrous Na₂SO₄. The solvent was rotovapped off and to the residue wereadded NaBr (0.12 g, 1.17 mmol) and DMF (10 mL). The suspension washeated up to 80° C. under N₂ for 2 hours. DMF was removed under vacuumand the residue was chromatographed on silica (EtOAc/hexanes 1:10) togive the desired product (0.191 g, 97% yield) as a pale yellow oil. ¹HNMR (CDCl₃, 300 MHz) δ 3.69-3.35 (series of multiplets, 13 H), 3.28-3.02(series of multiplets, 4 H), 2.18-2.04 (m, 3 H), 2.00-1.60 (series ofmultiplets, 16 H), 1.58-0.96 (series of multiplets, 11 H), 0.92 (d,J=6.34 Hz, 3H), 0.89 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ80.62, 79.81, 76.08, 65.07, 64.50, 64.34, 49.03, 48.98, 48.79, 46.49,46.46, 42.73, 42.02, 39.85, 35.47, 35.34, 35.12, 34.79, 34.72, 29.82,29.80, 29.74, 29.11, 27.91, 27.78, 27.69, 23.55, 23.07, 22.88, 18.10,12.62; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M−H]⁺) 706.3609(63.1%), calcd. 706.3591; 704.3616 (52.8%), calcd. 704.3611.

Compound 49: Compound 48 (0.191 g, 0.269 mmol) and 23 (0.295 g, 0.459mmol) was dissolved in DMF (3 mL, distilled over BaO at 6 mm Hg beforeuse) followed by the addition of NaH (0.054 g, 60% in mineral oil). Thesuspension was stirred under N₂ at room temperature for 24 hours. H₂O(100 mL) was added to quench excess NaH and the mixture was thenextracted with Et₂O (40 mL, 3×20 mL) and the combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. The desired product(0.177 g, 52% yield based on compound 23) was obtained as a pale yellowoil after SiO₂ chromatography (EtOAc/hexanes 1:6, then 1:2). IR (neat)2940, 2862, 2095, 1472, 1456, 1362, 1263, 1113 cm⁻¹; ¹H NMR(CDCl₃, 300MHz) δ 3.68-3.35 (series of multiplets, 26 H), 3.28-3.02 (series ofmultiplets, 8 H), 2.20-2.04 (m, 6 H), 1.96-1.60 (series of multiplets,30 H), 1.52-0.98 (series of multiplets, 12 H), 0.91 (d, J=6.59 Hz, 6 H),0.89 (s, 6 H), 0.65 (s, 6 H); ¹³C NMR(CDCl₃, 75 MHz) δ 80.68, 79.83,76.13, 71.71, 65.06, 64.48, 64.39, 49.08, 48.98, 48.80, 46.64, 46.44,42.71, 42.04, 39.88, 35.73, 35.49, 35.36, 35.14, 32.41, 29.84, 29.81,29.76, 29.14, 27.92, 27.78, 27.69, 26.58, 23.59, 23.08, 22.92, 18.12,12.64.

Compound 12: Compound 49 (0.219 g, 0.173 mmol) was dissolved in dry THF(10 mL) followed by the addition of LiAlH₄ (65 mg, 1.73 mmol). The greysuspension was stirred under N₂ at room temperature for 12 hours.Na₂SO₄.10H₂O powder was carefully added. After the grey color in thesuspension disappeared, anhydrous Na₂SO₄ was added and the precipitatewas filtered out. After the removal of solvent, the residue was purifiedby column chromatography (silica gel, MeOH/CH₂Cl₂/28% NH₃H₂O 2.5:2.5:1).After most of the solvent was rotavapped off from the fractionscollected, 5% HCl solution (2 mL) was added to dissolve the milkyresidue. The resulted clear solution was then extracted with Et₂O (2×10mL). 20% NaOH solution was then added until the solution became stronglybasic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄ and removal ofsolvent gave the desired product (0.147 g, 76% yield) as a white glass.IR (neat) 3364, 3287, 2934, 2861, 1596, 1464, 1363, 1105 cm⁻¹; ¹H NMR(20% CDCl₃ in CD₃OD, 500 MHz) δ 4.74 (bs, 12 H), 3.75-3.70 (m, 2 H),3.65-3.61 (m, 2 H), 3.57-3.52 (m, 6 H), 3.40 (t, J=3.60 Hz, 4 H), 3.30(bs, 4 H), 3.16-3.10 (m, 4 H), 2.84-2.73 (m, 12 H), 2.18-2.07 (m, 6 H),1.97-1.61 (series of multiplets, 30 H), 1.58-0.98 (series of multiplets,24 H), 0.95 (d, J=6.84 Hz, 6 H), 0.94 (s, 6 H), 0.70 (s, 6 H); ¹³C NMR(20% CDCl₃ in CD₃OD, 125 MHz) δ 81.70, 80.52, 77.09, 72.34, 67.75 (2C's), 67.07, 47.80, 47.13, 43.76, 42.87, 41.20, 40.65, 40.58, 40.14,36.43, 36.25, 36.08, 35.77, 34.15, 33.87 (2 C's), 33.18, 29.55, 28.92,28.47, 28.42, 27.25, 24.27, 23.54, 23.41, 18.70, 13.07; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 1113.9625 (68.8%), calcd.1113.9610.

Example 7 Syntheses of Compounds 111-113 and 116 a-d

Compounds 116a-d: Representative procedure: preparation of 116b. NaH(0.06 g, 60% in mineral oil, 1.49 mmol) and propyl bromide (0.136 mL,1.49 mmol) were added to a DMF solution of compound 23 (described in Liet al., J. Am. Chem. Soc. 1998, 120, 2961) (0.096 g, 0.149 mmol). Thesuspension was stirred under N₂ for 24 hr. H₂O (20 mL) was added, andthe mixture was extracted with hexanes (3×10 mL). The combined extractswere dried over Na₂SO₄ and concentrated in vacuo. Silica gelchromatography (10% EtOAc in hexanes) afforded the desired product (92mg, 90% yield) as a pale yellow oil. ¹H NMR (CDCl₃, 500 MHz) δ 3.68-3.64(m, 1 H), 3.61-3.57 (m, 1 H), 3.52 (t, J=6.1 Hz, 2 H), 3.49 (bs, 1 H),3.46-3.35 (m, 10 H), 3.25 (d, J=2.4 Hz, 1 H), 3.23-3.19 (m, 1 H),3.16-3.11 (m, 1 H), 3.09-3.03 (m, 1 H), 2.17-2.03 (m, 3 H), 1.95-1.55(m, 17 H), 1.51-1.40 (m, 4 H), 1.38-1.17 (m, 5 H), 1.11-0.96 (m, 3 H),0.93-0.89 (m, 9 H), 0.65 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.64,79.79, 76.08, 72.67, 71.59, 65.01, 64.44, 64.33, 49.04, 48.94, 48.75,46.61, 46.40, 42.68, 42.00, 39.83, 35.72, 35.45, 35.30, 35.10, 32.38,29.81, 29.77, 29.72, 29.09, 27.88, 27.76, 27.65, 26.52, 23.55, 23.12,23.04, 22.87, 18.06, 12.60, 10.79; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+Na]⁺) 708.4910 (23.5%), calcd. 708.4920.

Compounds 111-113: Representative procedure: preparation of 112.Compound 116b (0.092 g, 0.134 mmol) was dissolved in THF (10 mL)followed by the addition of LiAlH₄ (0.031 g, 0.81 mmol). The suspensionwas stirred under N₂ for 12 hr. Na₂SO₄.10H₂O (˜1 g) was then carefullyadded. After the gray color in the suspension dissipated, anhydrousNa₂SO₄ was added, and the precipitate was removed by filtration.Concentration and silica gel chromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O12:6:1, then 10:5:1) yielded a glass which was dissolved in 1 M HCl (2mL). The resulting clear solution was washed with Et₂O (2×10 mL). 20%NaOH solution was added to the aqueous phase until the solution becamestrongly basic. CH₂Cl₂ (3×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄ and concentratedin vacuo to give the desired product (0.045 g, 55% yield) as a whiteglass. 112: ¹H NMR (˜20% CDCl₃ in CD₃OD, 500 MHz) δ 4.73 (bs, 6 H),3.74-3.70 (m, 1 H), 3.65-3.61 (m, 1 H), 3.55 (t, J=6.3 Hz, 2 H),3.42-3.38 (m, 4 H), 3.33-3.30 (m, 2 H), 3.16-3.10 (m, 2 H), 2.83-2.73(m, 6 H), 2.18-2.06 (m, 3 H), 1.96-1.20 (series of multiplets, 26 H),1.12-0.98 (m, 3 H), 0.95-0.92 (m, 9 H), 0.70 (s, 3 H); ¹³C NMR (˜20%CDCl₃ in CD₃OD, 75 MHz) δ 81.67, 80.49, 77.04, 73.44, 72.28, 67.77,67.71, 67.06, 47.74, 47.08, 43.75, 42.82, 41.21, 40.60, 40.56, 40.12,36.47, 36.19, 36.04, 35.74, 34.09, 33.82, 33.78, 33.16, 29.49, 28.87,28.43, 27.18, 24.22, 23.66, 23.49, 23.40, 18.64, 13.04, 11.03; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 608.5348 (100%), calcd.608.5330. 111: ¹H NMR (˜20% CDCl₃ in CD₃OD, 500 MHz) δ 4.79 (bs, 6 H),3.74-3.71 (m, 1 H), 3.66-3.62 (m, 1 H), 3.55 (t, J=6.1 Hz, 2 H), 3.52(bs, 1 H), 3.38-3.28 (series of multiplets, 4 H), 3.33 (s, 3 H),3.16-3.10 (m, 2H), 2.83-2.72 (m, 6 H), 2.19-2.07 (m, 3 H), 1.97-1.62(series of multiplets, 15 H), 1.58-1.20 (series of multiplets, 9 H),1.13-0.98 (m, 3 H), 0.95 (d, J=6.3 Hz, 3 H), 0.93 (s, 3 H), 0.70 (s, 3H); ¹³C NMR (˜20% CDCl₃ in CD₃OD, 75 MHz) δ 81.82, 80.65, 77.20, 74.43,67.85, 67.18, 58.90, 47.80, 47.22, 43.91, 43.01, 41.31, 40.78, 40.69,40.22, 36.63, 36.35, 36.18, 35.86, 34.27, 33.97, 33.26, 29.60, 29.03,28.58, 28.53, 27.14, 24.33, 23.61, 23.45, 18.68, 13.06; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 602.4855 (100%), calcd.602.4873. 113: ¹H NMR (˜50% CDCl₃ in CD₃OD, 500 MHz) δ 4.08 (bs, 6 H),3.71-3.67 (m, 1 H), 3.62-3.58 (m, 1 H), 3.53 (t, J=6.3 Hz, 2 H), 3.49(bs, 1 H), 3.43-3.38 (m, 4 H), 3.31-3.27 (m, 2 H), 3.14-3.07 (m, 2 H),2.83-2.73 (m, 6 H), 2.16-2.03 (m, 3 H), 1.93-1.17 (series of multiplets,30 H), 1.10-0.96 (m, 3 H), 0.93-0.89 (m, 9 H), 0.67 (s, 3 H); ¹³C NMR(˜50% CDCl₃ in CD₃OD, 75 MHz) δ 80.51, 79.35, 75.85, 71.29, 70.83,66.73, 66.62, 65.96, 46.68, 45.98, 42.59, 41.63, 40.20, 39.53, 39.43,39.21, 35.34, 35.04, 35.00, 34.71, 33.11, 32.90, 32.82, 32.00, 29.15,28.49, 28.15, 27.75, 27.35, 26.22, 23.18, 22.60, 22.45, 22.34, 17.77,13.75, 12.22; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 636.5679(100%), calcd. 636.5669.

Example 8 Syntheses of Compounds 106 and 124

Compound 124: Compound 47 (0.256 g, 0.489 mmol) was dissolved in CH₂Cl₂(10 mL), and cooled to 0° C. followed by the addition of Na₂HPO₄ (0.69g, 4.89 mmol) and urea-hydrogen peroxide complex (UHP) (0.069 g, 0.733mmol). Trifluoroacetic anhydride (TFAA) (0.138 mL, 0.977 mmol) was thenadded dropwise. The suspension was stirred for 12 hr, and additional UHP(23 mg, 0.25 mmol) and TFAA (0.069 mL, 0.49 mmol) were added. Afteranother 12 hr, H₂O (30 mL) was added, and the resulting mixture wasextracted with EtOAc (3×20 mL). The combined extracts were washed withbrine (50 mL), dried over anhydrous Na₂SO₄, and concentrated in vacuo.SiO₂ chromatography (EtOAc/hexanes 1:5) afforded the desired product(0.145 g, 55% yield) as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 5.21(dd, J=9.3 and 7.3 Hz, 1 H), 3.70-3.57 (m, 2 H), 3.55 (t, J=6.0 Hz, 2H), 3.43-3.37 (m, 6 H), 3.32-3.25 (m, 3 H), 3.17-3.02 (m, 2 H),2.28-2.05 (m, 4 H), 2.03 (s, 3 H), 1.86-1.19 (series of multiplets, 19H), 0.97 (dd, J=14.5 and 3.3 Hz, 1 H), 0.90 (s, 3 H), 0.78 (s, 3 H); ¹³CNMR (CDCl₃, 75 MHz) δ 171.08, 79.71, 78.03, 75.72, 75.53, 65.41, 65.04,64.53, 48.79, 48.70, 46.49, 41.92, 39.44, 37.81, 35.45, 35.22, 35.10,29.73, 29.63, 28.89, 28.33, 27.50, 27.34, 23.39, 22.97, 22.92, 21.28,12.72; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M−H]⁺) 614.3798(24.5%), calcd. 614.3778.

Compound 106: Compound 124 (0.145 g, 0.236 mmol) was dissolved in CH₂Cl₂(2 mL) and MeOH (1 mL). 20% NaOH solution (0.2 mL) was added. Themixture was stirred for 12 hr, and anhydrous Na₂SO₄ was used to removewater. After concentration in vacuo, the residue was purified by silicagel chromatography (EtOAc/hexanes 1:3) to afford the desired product(0.124 g, 92% yield) as a colorless oil. ¹H NMR (CDCl₃, 300 MHz) δ 4.29(bs, 1 H), 3.69-3.60 (m, 2 H), 3.52 (t, J=6.0 Hz, 2 H), 3.45-3.32 (m, 8H), 3.26 (d, J=2.7 Hz, 1 H), 3.17-3.02 (m, 2 H), 2.19-1.94 (m, 4 H),1.90-1.62 (series of multiplets, 13 H), 1.57-1.20 (series of multiplets,7 H), 0.97 (dd, J=14.3 and 3.1 Hz, 1H), 0.90 (s, 3 H), 0.73 (s, 3 H);¹³C NMR (CDCl₃, 75 MHz) δ 79.69, 78.03, 75.47, 73.38, 65.46, 65.00,64.47, 48.87, 48.68, 46.83, 41.93, 39.71, 37.87, 35.43, 35.20, 35.09,29.96, 29.69, 29.59, 29.53, 28.89, 28.44, 27.48, 23.72, 22.91, 22.71,11.77. The alcohol (0.124 g, 0.216 mmol) was dissolved in dry THF (20mL) followed by the addition of LiAlH₄ (33 mg, 0.866 mmol). The graysuspension was stirred under N₂ for 12 hr. Na₂SO₄.10H₂O (˜2 g) wascarefully added. After the gray color in the suspension dissipated,anhydrous Na₂SO₄ was added and the precipitate was removed byfiltration. After the removal of solvent, the residue was purified bycolumn chromatography (SiO₂, MeOH/CH₂Cl₂/28% NH₃.H₂O 2.5:2.5:1). Afterconcentration of the relevant fractions, 1 M HCl (2 mL) was added todissolve the milky residue. The resulting clear solution was washed withEt₂O (2×10 mL). To the aqueous phase, 20% NaOH solution was added untilthe solution became strongly basic. CH₂Cl₂ (20 mL, 2×10 mL) was used toextract the basic solution. The combined extracts were dried overanhydrous Na₂SO₄ and removal of solvent gave the desired product (0.050g, 47% yield) as a colorless oil. ¹H NMR (20% CDCl₃ in CD₃OD, 300 MHz) δ4.77 (s, 7 H), 4.25 (t, J=8.5 Hz, 1 H), 3.75-3.68 (m, 1 H), 3.66-3.58(m, 1 H), 3.55 (t, J=6.1 Hz, 2 H), 3.48-3.41 (m, 1 H), 3.34 (bs, 1 H),3.30 (d, J=3.6 Hz, 1 H), 3.17-3.08 (m, 2 H), 2.86-2.70 (m, 6H),2.20-1.91 (m, 4 H), 1.88-1.16 (series of multiplets, 19 H), 1.00 (dd,J-=14.2 and 3.0 Hz, 1 H), 0.93 (s, 3 H), 0.73 (s, 3 H); ¹³C NMR (20%CDCl₃ in CD₃OD, 75 MHz) δ 80.62, 79.12, 76.74, 73.77, 68.50, 67.79,67.17, 47.69, 43.04, 40.76, 40.64, 40.62, 40.22, 39.01, 36.32, 36.25,35.94, 34.27, 33.97, 33.72, 30.13, 29.53, 28.43, 24.48, 23.58, 23.40,12.38; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 496.4108 (100%),calcd. 496.4114.

Example 9 Syntheses of Compounds 109, and 126-129

Compound 126: Compound 125 (2.30 g, 3.52 mmol) was dissolved in MeOH (50mL) and CH₂Cl₂ (100 mL). A small amount of Et₃N was added, and thesolution was cooled to −78° C. Ozone was bubbled through the solutionuntil a blue color persisted. Me₂S (4 mL) was introduced followed by theaddition of NaBH₄ (0.266 g, 0.703 mmol) in MeOH (10 mL). The resultingsolution was allowed to warm and stir overnight. The solution wasconcentrated in vacuo, and brine (60 mL) was added. The mixture wasextracted with EtOAc (40 ml, 2×30 mL), and the combined extracts werewashed with brine and dried over anhydrous Na₂SO₄. Silica gelchromatography (EtOAc) afforded the product (1.24 g, 76% yield) as awhite solid. m.p. 219-220° C.; ¹H NMR (CDCl₃, 300 MHz) δ 5.10 (t, J=2.8Hz, 1 H), 4.90 (d, J=2.7 Hz, 1 H), 3.73-3.59 (m, 2 H), 3.56-3.44 (m, 1H), 2.13 (s, 3 H), 2.09 (s, 3 H), 2.07-0.95 (series of multiplets, 23H), 0.91 (s, 3 H), 0.83 (d, J=6.3 Hz, 3 H), 0.74 (s, 3 H); ¹³C NMR(CDCl₃, 75 MHz) δ 170.84, 170.82, 75.63, 71.77, 71.03, 60.73, 48.10,45.26, 43.54, 41.16, 38.78, 37.89, 35.00, 34.43, 32.26, 31.50, 30.60,29.07, 27.50, 25.70, 22.96, 22.71, 21.81, 21.63, 18.18, 12.35; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 465.3197 (20%), calcd. 465.3216.

Compound 127: Compound 126 (1.24 g, 2.67 mmol) was dissolved in MeOH (30mL), and NaOH (0.54 g, 13.4 mmol) was added. The suspension was refluxedunder N₂ for 24 hr. The MeOH was removed in vacuo followed by theaddition of H₂O (50 mL). The precipitate was filtered, washed with H₂Oand then dried in vacuo to give a white solid (1.02 g). This solid wasdissolved in DMF (40 mL) followed by the sequential addition of NEt₃(1.12 mL, 8.02 mmol), DMAP (16.3 mg, 0.13 mmol) and trityl chloride(1.49 g, 5.34 mmol). The suspension was stirred under N₂ for 12 hr andthen heated up to 50° C. for 24 hr. H₂O (100 mL) was added to the cooledsuspension, and the mixture was extracted with EtOAc (3×50 mL). Thecombined extracts were washed with brine (100 mL), dried over anhydrousNa₂SO₄, and concentrated in vacuo. Silica gel chromatography (EtOAc)afforded the product (1.20 g, 72% yield) as a pale yellow glass. To thisglass was added dry THF (80 mL) and NaH (60% in mineral oil, 0.77 g,19.3 mmol). The suspension was refluxed under N₂ for half an hour beforethe introduction of allylbromide (1.67 mL, 19.3 mmol). After 48 hr atreflux, another 10 eq. of NaH and allylbromide were introduced. Afteranother 48 hr, the reaction mixture was cooled and H₂O (100 mL) wasslowly added. The resulting mixture was extracted with hexanes (3×50mL), and the combined extracts were washed with brine (100 mL) and driedover anhydrous Na₂SO₄. Silica gel chromatography (5% EtOAc in hexanes)afforded the product (1.27 g, 64% yield for all three steps) as a clearglass. ¹H NMR (CDCl₃, 300 MHz) δ 7.46-7.43 (m, 6 H), 7.29-7.16 (m, 9 H),5.98-5.81 (m, 3 H), 5.29-5.18 (m, 3 H), 5.14-5.03 (m, 3 H), 4.11-3.97(m, 4 H), 3.75-3.67 (m, 2 H), 3.49 (bs, 1 H), 3.32-3.13 (d, J=2.4 Hz, 1H), 3.20-3.13 (m, 2 H), 3.00 (m, 1 H), 2.33-2.12 (m, 3 H), 2.03-0.92(series of multiplets, 19 H), 0.88 (s, 3 H), 0.78 (d, J=6.6 Hz, 3 H),0.65 (s, 3 H); C NMR (CDCl₃, 75 MHz) δ 144.71, 136.08, 136.04, 135.94,128.80, 127.76, 126.86, 116.30, 115.57, 86.53, 80.77, 79.20, 74.96,69.42, 69.34, 68.81, 62.00, 46.87, 46.48, 42.67, 42.11, 39.90, 36.15,35.50, 35.14, 35.10, 33.23, 28.99, 28.09, 27.75, 27.56, 23.36, 23.32,23.12, 18.24, 12.66; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)765.4875 (100%), calcd. 765.4859.

Compound 128: To a THF (40 mL) solution of 127 (1.27 g, 1.71 mmol) wasadded 9-BBN (0.5 M solution in THF, 17.1 mL). The mixture was stirredfor 12 hr before the addition of NaOH (20% solution, 10 mL) and H₂O₂(30% solution, 10 mL). The resulted mixture was refluxed for 1 hrfollowed by the addition of brine (100 mL) and extraction with EtOAc(4×30 mL). The combined extracts were dried over anhydrous Na₂SO₄ andconcentrated in vacuo. Silica gel chromatography (5% MeOH in CH₂Cl₂)afforded the product (1.26 g, 93% yield) as a clear glass. ¹H NMR (5%CD₃OD in CDCl₃, 300 MHz) δ 7.46-7.43 (m, 6 H), 7.32-7.20 (m, 9 H), 3.94(s, 3 H), 3.78-3.56 (m, 10 H), 3.48 (bs, 1 H), 3.32-3.26 (m, 2 H),3.24-3.12 (m, 3 H), 3.00 (dd, J=8.2 and 6.1 Hz, 1 H), 2.23-1.96 (m, 3H), 1.90-0.95 (series of multiplets, 25 H), 0.90 (s, 3 H), 0.77 (d,J=6.6 Hz, 3 H), 0.66 (s, 3 H); ¹³C NMR (5% CD₃OD in CDCl₃, 75 MHz) δ144.52, 128.64, 127.64, 126.76, 86.43, 80.55, 79.31, 77.65, 77.23,76.80, 76.06, 66.17, 66.01, 65.41, 61.93, 61.20, 60.73, 60.39, 47.29,46.08, 42.65, 41.62, 39.49, 36.02, 35.10, 34.89, 34.77, 32.89, 32.71,32.41, 32.26, 28.68, 27.70, 27.51, 27.19, 23.26, 22.66, 22.50, 18.23,12.34; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 819.5169(100%), calcd. 819.5099.

Compound 129: To a CH₂Cl₂ (50 mL) solution of compound 128 (1.26 g, 1.58mmol) at 0° C. was added Et₃N (0.92 mL, 6.60 mmol) followed by mesylchloride (0.47 mL, 6.05 mmol). After 15 minutes, H₂O (10 mL) wasfollowed by brine (80 mL). The mixture was extracted with EtOAc (60 mL,2×30 mL) and the combined extracts were dried over anhydrous Na₂SO₄.After removal of solvent in vacuo, the residue was dissolved in DMSO (10mL) and NaN₃ (1.192 g, 18.3 mmol) was added. The suspension was heatedto 60° C. under N₂ overnight. H₂O (100 mL) was added, and the mixturewas extracted with EtOAc (3×40 mL). The combined extracts were washedwith brine and dried over anhydrous Na₂SO₄. Removal of the solvent invacuo afforded a pale yellow oil. The oil was dissolved in MeOH (10 mL)and CH₂Cl₂ (20 mL) and TsOH (17.4 mg, 0.092 mmol) was added. After 12hr, saturated aqueous NaHCO₃ (20 mL) and brine (50 mL) were added andthe mixture was extracted with EtOAc (3×40 mL). The combined extractswere washed with brine (50 mL) and dried over anhydrous Na₂SO₄. Silicagel chromatography (EtOAc/hexanes 1:3) afforded the desired product(0.934, 94%) as a pale yellow oil. ¹H NMR (CDCl₃, 500 MHz) δ 3.75-3.70(m, 1 H), 3.68-3.63 (m, 2 H), 3.62-3.57 (m, 1 H), 3.53 (t, J=6.1 Hz, 2H), 3.50 (bs, 1 H), 3.46-3.38 (m, 6 H), 3.26 (d, J=2.4 Hz, 1 H),3.24-3.20 (m, 1 H), 3.16-3.12 (m, 1 H), 3.10-3.04 (m, 1 H), 2.17-2.04(m, 3 H), 1.96-1.63 (m, 14 H), 1.53-1.45 (m, 3 H), 1.35-1.20 (m, 7 H),1.08-1.00 (m, 1H), 0.97-0.88 (m, 1 H), 0.94 (d, J=6.8 Hz, 3 H), 0.89 (s,3 H), 0.67 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 80.64, 79.81, 76.06,65.05, 64.49, 64.34, 61.03, 49.02, 48.98, 48.78, 46.93, 46.53, 42.76,42.01, 39.83, 39.14, 35.46, 35.33, 35.12, 32.97, 29.79, 29.73, 29.10,27.90, 27.68, 23.56, 23.06, 22.88, 18.24, 12.60; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 652.4285 (100%), calcd.652.4295.

Compound 109: Compound 129 (0.245 g, 0.391 mmol) was dissolved in THF(30 mL) followed by the addition of LiAlH₄ (59 mg, 1.56 mmol). The graysuspension was stirred under N₂ 12 hr. Na₂SO₄. 10H₂O powder (˜1 g) wascarefully added. After the gray color in the suspension dissipated,anhydrous Na₂SO₄ was added and the precipitate was removed byfiltration. After the removal of solvent, the residue was purified bysilica gel chromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O 10:5:1 then10:5:1.5). The solvent was removed from relevant fractions, and 1 M HCl(4 mL) was added to dissolve the residue. The resulting clear solutionwas extracted with Et₂O (3×10 mL). 20% NaOH solution was added until thesolution became strongly basic. CH₂Cl₂ (4×10 mL) was used to extract thebasic solution. The combined extracts were dried over anhydrous Na₂SO₄,and removal of solvent in vacuo gave the desired product (0.15 g, 71%yield) as a colorless oil. ¹H NMR (˜20% CD₃OD in CDCl₃, 500 MHz) δ 4.73(bs, 7 H), 3.74-3.70 (m, 1 H), 3.65-3.60 (m, 2 H), 3.56-3.52 (m, 4 H),3.31-3.28 (m, 2 H), 3.16-3.09 (m, 2H), 2.82-2.71 (m, 6 H), 2.19-2.06 (m,3 H), 1.97-1.66 (series of multiplets, 15 H), 1.58-1.48 (m, 3 H),1.38-0.98 (m, 7 H), 0.96 (d, J=6.8 Hz, 3 H), 0.93 (s, 3 H), 0.71 (s, 3H); ¹³C NMR (˜20% CD₃OD in CDCl₃, 75 MHz) δ 81.80, 80.60, 77.17, 67.88,67.86, 67.18, 60.73, 48.11, 47.28, 43.93, 42.99, 41.34, 40.76, 40.72,40.24, 39.70, 36.33, 36.18, 35.86, 34.29, 33.99, 33.96, 33.83, 29.60,29.00, 28.57, 28.54, 24.33, 23.59, 23.48, 18.86, 13.04; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 552.4756 (100%), calcd.552.4772.

Example 10 Syntheses of Compounds 108 and 130

Compound 130: o-NO₂C₆H₄SeCN (0.094 g, 0.21 mmol) and Bu₃P (0.095 mL,0.38 mmol) were stirred in dry THF (5 mL) at 0° C. for ½ hr followed bythe addition of compound 129 (0.10 g, 0.159 mmol) in THF (2 mL). Thesuspension was stirred for 1 hr followed by the addition of H₂O₂ (30%aqueous solution, 2 mL). The mixture was stirred for 12 hr followed byextraction with hexanes (4×10 mL). The combined extracts were dried overanhydrous Na₂SO₄. The desired product (0.035 g, 36% yield) was obtainedas pale yellowish oil after silical gel chromatography (10%EtOAc/hexanes). ¹H NMR (CDCl₃, 500 MHz) δ 5.73-5.66 (ddd, J=17.1, 10.2,8.3 Hz, 1 H), 4.90 (dd, J=17.1, 2.0 Hz, 1 H), 4.82 (dd, J=10.2 Hz, 1.96Hz, 1 H), 3.68-3.64 (m, 1 H), 3.62-3.58 (m, 1 H), 3.54-3.26 (m, 9 H),3.25-3.22 (m, 2 H), 3.15-3.11 (m, 1 H), 3.10-3.04 (m, 1 H), 2.17-1.62(series of multiplets, 18 H), 1.51-1.43 (m, 2 H), 1.35-1.18 (m, 4 H),1.06-0.91 (m, 2 H), 1.02 (d, J=6.3 Hz, 3 H), 0.90 (s, 3 H), 0.68 (s, 3H); ¹³C NMR (CDCl₃, 75 MHz) δ 145.50, 111.72, 80.60, 79.82, 76.09,65.06, 64.50, 64.45, 49.05, 48.97, 48.79, 46.43, 46.13, 42.76, 42.03,41.30, 39.84, 35.49, 35.34, 35.15, 29.82, 29.80, 29.75, 29.11, 28.00,27.84, 27.68, 23.56, 23.08, 22.95, 19.79, 12.87; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 634.4167 (90.6%), calcd.634.4169.

Compound 108: Compound 130 (0.105 g, 0.172 mmol) was dissolved in CH₂Cl₂(5 mL) and MeOH (5 mL) at −78° C. 03 was bubbled into the solution forca. 20 min. Me₂S (1 mL) was added followed, and the solvent was removedin vacuo. The residue was dissolved in THF (15 mL), and LiAlH₄ (0.033 g,0.86 mmol) was-added. The suspension was stirred for 12 hr. Na₂SO₄.10H₂O (˜2 g) was carefully added. After the gray color of the suspensiondissipated, anhydrous Na₂SO₄ was added and the precipitate was removedby filtration. Concentration and silica gel chromatography(CH₂Cl₂/MeOH/28% NH₃.H₂O 10:5:1.5 then 9:6:1.8) yielded a white glass.To this material was added 1 M HCl (4 mL). The resulting clear solutionwas washed with Et₂O (3×10 mL). 20% NaOH solution was added to theaqueous phase until the solution became strongly basic. CH₂Cl₂ (4×10 mL)was used to extract the basic solution. The combined extracts were driedover anhydrous Na₂SO₄ and removal of solvent gave the desired product(0.063 g, 68% yield) as a colorless oil. ¹H NMR (˜10% CD₃OD in CDCl₃,500 MHz) δ 4.76 (bs, 7 H), 3.75-3.71 (m, 1 H), 3.66-3.62 (m, 1 H),3.58-3.52 (in, 4 H), 3.33-3.29 (m, 2 H), 3.22 (dd, J=10.5 and 7.6 Hz, 1H), 3.15-3.09 (m, 2 H), 2.81 (t, J=6.8 Hz, 2 H), 2.76-2.71 (m, 4 H),2.19-2.08 (m, 3 H), 2.00-1.66 (series of multiplets, 14 H), 1.58-1.45(m, 3 H), 1.40-1.08 (m, 5 H), 1.03 (d, J=6.8 Hz, 3 H), 1.02-0.96 (m, 1H), 0.93 (s, 3 H), 0.72 (s, 3 H); ¹³C NMR (˜10% CD₃OD in CDCl₃, 75 MHz)δ 81.74, 80.64, 77.23, 67.95, 67.87, 67.18, 47.32, 44.59, 43.72, 43.01,41.26, 40.80, 40.71, 40.23, 40.02, 36.36, 36.20, 35.87, 34.27, 33.99,33.90, 29.60, 29.05, 28.58, 28.08, 24.49, 23.62, 23.46, 16.84, 13.12;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 538.4578 (4.7%), calcd.538.4584.

Example 11 Syntheses of Compounds 132-135

Compound 132: Compound 115 (0.118 g, 0.183 mmol) was dissolved in dryCH₂Cl₂ (10 mL), and SO₃ pyridine complex (0.035 g, 0.22 mmol) was added.The suspension was stirred for 12 hr. The solvent was removed in vacuoto give white powder. To the white powder was added 1 M HCl (10 mL) andthe resulting mixture was extracted with CH₂Cl₂ (4×10 mL). The combinedextracts were dried over anhydrous Na₂SO₄. The desired product (0.11 g,84%) was obtained as a pale yellow oil after silica gel chromatography(10% MeOH in CH₂Cl₂). ¹H NMR (˜10% CD₃OD in CDCl₃, 500 MHz) δ 4.03 (t,J=6.8 Hz, 2 H), 3.69-3.65 (m, 1 H), 3.62-3.58 (m, 1 H), 3.55 (t, J=6.1Hz, 2 H), 3.51 (bs, 1 H), 3.46-3.38 (m, 6 H), 3.27 (d, J=2.4 Hz, 1 H),3.26-3.21 (m, 1H), 3.18-3.07 (m, 2 H), 2.18-2.03 (m, 3 H), 1.95-1.47(series of multiplets, 19 H), 1.40-0.96 (series of multiplets, 9 H),0.92 (d, J=6.8 Hz, 3 H), 0.91 (s, 3 H), 0.66 (s, 3, H); ¹³C NMR (˜10%CD₃OD in CDCl₃, 75 MHz) δ 80.43, 79.68, 75.87, 69.30, 64.82, 64.32,64.14, 48.78, 48.73, 48.50, 46.44, 46.21, 42.49, 41.76, 39.61, 35.36,35.17, 35.06, 34.85, 31.73, 29.53, 29.46, 29.44, 28.84, 27.68, 27.48,27.38, 25.91, 23.30, 22.75, 22.66, 17.70, 12.32; HRFAB-MS(thioglycerol+Na³⁰ matrix) m/e: ([M−H+2Na]⁺) 768.3831 (100%), calcd.768.3843. The azides were reduced by treating the triazide (0.11 g, 0.15mmol) with Ph₃P (0.20 g, 0.77 mmol) in THF (10 mL) and H₂O (1 mL). Themixture was stirred for 3 days. The solvent was removed in vacuo, andthe residue was purified by silica gel chromatography (CH₂Cl₂/MeOH/28%NH₃.H₂O 12:6:1 then 10:5:1.5) to afford the desired product (0.077 g,78% yield) as a glass. HCl in Et₂O (1 M, 0.5 mL) was added to the glassto give the corresponding HCl salt. ¹H NMR (˜10% CDCl₃ in CD₃OD, 500MHz) δ 4.81 (s, 10 H), 4.07-3.97 (m, 2 H), 3.82 (bs, 1 H), 3.11 (bs, 1H), 3.65 (t, J=5.2 Hz, 2 H), 3.57 (bs, 1 H), 3.37-3.30 (m, 2 H),3.22-3.02 (m, 8 H), 2.12-1.71 (series of multiplets, 17 H), 1.65-1.01(series of multiplets, 13 H), 0.97 (d, J=6.8 Hz, 3 H), 0.94 (s, 3 H),0.73 (s, 3 H); ¹³C NMR (˜10% CDCl₃ in CD₃OD, 75 MHz) δ 81.89, 80.58,77.50, 70.04, 66.71, 66.56, 66.02, 47.11, 46.76, 44.20, 42.66, 40.50,39.60, 39.40, 36.24, 36.11, 35.89, 35.67, 32.28, 29.38, 29.23, 29.10,28.94, 28.49, 26.06, 24.21, 23.46, 23.30, 18.50, 12.86; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 668.4271 (100%), calcd.668.4258.

Compound 133: The mesylate derived from 23 (0.19 g, 0.264 mmol) wasstirred with excess octyl amine (2 mL) at 80° C. for 12 hr. Afterremoval of octylamine in vacuo, the residue was chromatographed (silicagel, EtOAc/hexanes 1:4 with 2% Et₃N) to afford the desired product (0.19g, 95% yield) as a pale yellow oil. ¹H NMR (CDCl₃, 300 MHz)δ_(—)3.69-3.37 (series of multiplets, 11 H), 3.26-3.00 (m, 4 H),2.61-2.53 (m, 4H), 2.20-2.02 (m, 3 H), 1.98-0.99 (series of multiplets,40 H), 0.92-0.85 (m, 9 H), 0.65 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ80.60, 79.74, 76.05, 64.97, 64.40, 64.28, 50.79, 50.25, 49.00, 48.90,48.71, 46.47, 46.34, 42.65, 41.96, 39.80, 35.77, 35.41, 35.27, 35.05,33.73, 31.96, 30.25, 29.76, 29.74, 29.67, 29.39, 29.05, 27.84, 27.61,27.55, 26.70, 23.50, 23.00, 22.82, 22.79, 18.06, 14.23, 12.54; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 755.6012 (100%), calcd.755.6024. The triazide (0.18 g, 0.239 mmol) was dissolved in THF (10 mL)and EtOH (10 mL). Lindlar catalyst (44 mg) was added, and the suspensionwas shaken under H₂ (50 psi) for 12 hr. After removal of the solvent invacuo, the residue was purified by silica gel chromatography(CH₂Cl₂/MeOH/28% NH₃.H₂O 10:5:1, then 10:5:1.5). To the product, 1 M HCl(2 mL) and the resulting clear solution was extracted with Et₂O (2×10mL). 20% NaOH solution was added until the solution became stronglybasic. CH₂Cl₂ (20 mL, 2×10 mL) was used to extract the basic solution.The combined extracts were dried over anhydrous Na₂SO₄, and removal ofsolvent in vacuo gave the desired product (0.114 g, 68% yield) as aclear oil. ¹H NMR (˜20% CDCl₃ in CD₃OD, 500 MHz) δ 4.79 (bs, 7 H),3.74-3.70 (m, 1 H), 3.66-3.61 (m, 1 H), 3.56-3.51 (m, 3 H), 3.31-3.29(m, 2 H), 3.16-3.09 (m, 2 H), 2.88-2.72 (m, 6 H), 2.59-2.51 (m, 4 H),2.18-2.07 (m, 3 H), 1.97-1.66 (series of multiplets, 14 H), 1.62-0.97(series of multiplets, 25 H), 0.95 (d, J=6.3 Hz, 3 H), 0.93 (s, 3 H),0.89 (t, J=6.8 Hz, 3H), 0.70 (s, 3 H); ¹³C NMR (˜20% CDCl₃ in CD₃OD, 75MHz) δ 81.82, 80.63, 77.23, 67.85, 67.19, 51.20, 50.69, 47.82, 47.24,43.92, 43.01, 41.30, 40.80, 40.68, 40.22, 36.74, 36.38, 36.20, 35.87,34.66, 34.15, 33.87, 32.90, 30.54, 30.39, 30.30, 29.64, 29.03, 28.59,28.41, 26.96, 24.37, 23.65, 23.48, 18.75, 14.63, 13.09; HRFAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M+H]⁺) 677.6309 (46.6%), calcd.677.6309.

Compound 134: Compound 133 (0.08 g, 0.12 mmol) was dissolved in CHCl₃ (5mL) and MeOH (5 mL), aminoiminosulfonic acid (0.045 g, 0.36 mmol) wasadded, and the suspension was stirred for 12 hr. The solvent was removedin vacuo, and the residue was dissolved in 1 M HCl (6 mL) and H₂O (10mL). The solution was washed with Et₂O (3×5 mL), and 20% NaOH solutionwas then added dropwise until the solution became strongly basic. Thebasic mixture was extracted with CH₂Cl₂ (4×5 mL). The combined extractswere dried over anhydrous Na₂SO₄ and concentrated in vacuo to give thedesired product (0.087 g, 91% yield) as a white glass. ¹H NMR (˜20%CDCl₃ in CD₃OD, 500 MHz) δ 4.96 (bs, 13 H), 3.74-3.68 (m, 1 H),3.65-3.50 (m, 4 H), 3.38-3.18 (series of multiplets, 10 H), 2.60-2.50(m, 4 H), 2.15-1.99 (m, 3 H), 1.88-1.72 (m, 14 H), 1.60-0.99 (series ofmultiplets, 25 H), 0.94 (bs, 6 H), 0.89 (t, J=6.6 Hz, 3 H), 0.71 (s, 3H); ¹³C NMR (˜20% CDCl₃ in CD₃OD, 75 MHz) δ 159.00, 158.87, 158.72,81.68, 79.93, 76.95, 66.59, 65.93, 65.45, 50.82, 50.40, 47.64, 46.94,43.67, 42.27, 40.18, 39.25, 36.19, 35.66, 35.40, 34.21, 32.45, 30.51,30.26, 30.18, 30.10, 29.86, 29.35, 28.71, 28.15, 28.00, 26.87, 23.94,23.44, 23.23, 23.12, 18.61, 14.42, 12.98; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+H]⁺) 803.6958 (18.4%), calcd. 803.6953.

Compound 135: The mesylate derived from 23 (0.092 g, 0.128 mmol) wasdissolved in DMSO (2 mL) followed by the addition of NaN₃ (0.0167 g,0.256 mmol). The suspension was heated to 70° C. for 12 hr. H₂O (20 mL)was added to the cooled suspension, and the mixture was extracted withEtOAc/hexanes (1:1) (20 mL, 3×10 mL). The combined extracts were washedwith brine (30 mL), dried over anhydrous Na₂SO₄, and concentrated invacuo to give the product (0.081 g, 95% yield) as a pale yellow oil. ¹HNMR (CDCl₃, 300 MHz) δ 3.69-3.36 (m, 11 H), 3.25-3.02 (m, 6 H),2.20-2.02 (m, 3 H), 1.97-1.60 (m, 15 H), 1.55-0.98 (m, 13 H), 0.92 (d,J=6.3 Hz, 3 H), 0.89 (s, 3 H), 0.66 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz)δ_(—)80.59, 79.77, 76.03, 65.01, 64.46, 64.30, 52.12, 48.99, 48.95,48.76, 46.44, 46.42, 42.70, 41.99, 39.82, 35.56, 35.44, 35.31, 35.09,33.09, 29.79, 29.77, 29.71, 29.08, 27.88, 27.78, 27.66, 25.65, 23.53,23.03, 22.85, 18.00, 12.58; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 691.4512 (100%), calcd. 691.4496. The tetraazide (0.081 g,0.12 mmol) was dissolved in THF (5 mL) and EtOH (10 mL). Lindlarcatalyst (30 mg) was added, and the suspension was shaken under H₂ (50psi) for 12 hr. After removal of the solvent in vacuo, the residue waspurified by silica gel chromatography (CH₂Cl₂/MeOH/28% NH₃.H₂O 5:3:1,then 2:2:1). To the product, 1 M HCl (2 mL) was added, and the resultingsolution was washed with Et₂O (2×10 mL). 20% NaOH solution was added tothe aqueous phase until the solution became strongly basic. CH₂Cl₂ (10mL, 2×5 mL) was used to extract the basic solution. The combinedextracts were dried over anhydrous Na₂SO₄, and concentration in vacuogave the desired product (0.044 g, 64% yield) as a colorless oil. ¹H NMR(˜20% CDCl₃ in CD₃OD, 500 MHz) δ 4.79 (bs, 8 H), 3.74-3.70 (m, 1 H),3.66-3.62 (m, 1 H), 3.56-3.52 (m, 3 H), 3.31-3.27 (m, 2 H), 3.16-3.10(m, 2 H), 2.82-2.70 (m, 6 H), 2.64-2.54 (m, 2 H), 2.19-2.07 (m, 3 H),1.99-1.66 (series of multiplets, 14 H), 1.58-0.96 (series of multiplets,13 H), 0.96 (d, J=6.6 Hz, 3 H), 0.93 (s, 3 H), 0.70 (s, 3 H); ¹³C NMR(˜20% CDCl₃ in CD₃OD, 75 MHz) δ 81.96, 90.76, 77.33, 67.92, 67.26,47.84, 47.33, 44.04, 43.24, 43.15, 41.40, 40.91, 40.78, 40.29, 36.82,36.48, 36.28, 35.96, 34.39, 34.11, 30.59, 29.69, 29.13, 28.68, 28.64,24.43, 23.69, 23.48, 18.77, 13.06; HRFAB-MS (thioglycerol+Na⁺ matrix)m/e: ([M+H]⁺) 565.5041 (100%), calcd. 565.5057.

Example 12 Syntheses of Compounds 203a-b, 207a-c, 208a-c, 209a-c, and210a-b

Compounds 203a-b, 207a-c, 208a-c, 209a-c, and 210a-b: BOC-glycine wasreacted with DCC, DMAP and cholic acid derivative 201 (Scheme 11) togive triester 202a in good yield. A similar reaction incorporatingBOC-β-alanine was also successful, giving 202b. Deprotection of 202a and202b with HCl in dioxane, followed by purification (SiO₂ chromatographywith a CH₂Cl₂MeOH/NH₄OH eluent), gave triesters 203a and 203b in goodyield.

Triamides of glycine and β-alanine (207a and 207b, respectively) wereformed using the same reaction conditions (Scheme 12). Triamides withα-branched amino acids could also be formed. For example, under theconditions described, a triamide with bis-BOC-lysine side chains wasformed (compound 207c). The C24 esters of 207a-c were hydrolyzed withLiOH in THF and methanol to give alcohols 208a-c. Deprotection using HClin dioxane (208a-c) gave triamides 209a-c in good yield. In addition,alcohols 208a and 208b were mesylated and reacted with benzylmethylamine. Deprotection of the resulting compounds with HCl in dioxane gavetriamides 210a and 210b (Scheme 12). The antibacterial properties ofthese compounds are summarized in Table 14.

Example 13 Synthesis of Compounds 302, 312-321, 324-326, 328-331 and341-343

Compound 302: Compound 308 (5β-cholanic acid 3,7,12-trione methyl ester)was prepared from methyl cholate and pyridinium dichromate in nearquantitative yield from methyl cholate. Compound 308 can also beprepared as described in Pearson et al., J. Chem. Soc. Perkins Trans. l1985, 267; Mitra et al., J. Org. Chem. 1968, 33, 175; and Takeda et al.,J. Biochem. (Tokyo) 1959, 46, 1313. Compound 308 was treated withhydroxylamine hydrochloride and sodium acetate in refluxing ethanol for12 hr (as described in Hsieh et al., Bioorg. Med. Chem. 1995, 3, 823),giving 309 in 97% yield.

A 250 ml three neck flask was charged with glyme (100 ml); to this wasadded 309 (1.00 g, 2.16 mmol) and sodium borohydride (2.11 g, 55.7mmol). TiCl₄ (4.0 mL, 36.4 mmol) was added to the mixture slowly undernitrogen at 0° C. The resulting green mixture was stirred at roomtemperature for 24 hours and then refluxed for another 12 h. The flaskwas cooled in an ice bath, and ammonium hydroxide (100 mL) was added.The resulting mixture was stirred for 6 hours at room temperature. Conc.HCl (60 mL) was added slowly, and the acidic mixture was stirred for 8hours. The resulting suspension was made alkaline by adding solid KOH.The suspension was filtered and the solids were washed with MeOH. Thecombined filtrate and washings were combined and concentrated in vacuo.The resulting solid was suspended in 6% aqueous KOH (100 mL) andextracted with CH₂Cl₂ (4×75 mL). The combined extracts were dried overNa₂SO₄, and solvent was removed in vacuo to give 1.14 g of a whitesolid. The mixture was chromatographed on silica gel (CH₂Cl₂/MeOH/NH₄OH12:6:1) giving 302 (0.282 g, 33% yield), 3 (0.066 g, 8% yield), 4 (0.118g, 14% yield).

Compound 302: m.p. 200-2020 C; ¹H NMR (˜10% CDCl₃ in CD₃OD, 300 MHz) δ4.81 (bs, 7 H), 3.57-3.49 (m, 2 H), 3.14 (t, J=3.2 Hz, 1 H), 2.97 (bs, 1H), 2.55-2.50 (m, 1 H), 2.15-2.10 (m, 1 H), 1.95-1.83 (m, 3 H),1.74-0.99 (series of multiplets, 20 H), 1.01 (d, J=6.4 Hz, 3 H), 0.95(s, 3 H), 0.79 (s, 3 H); ¹³C NMR (˜10% CDCl₃ in CD₃OD, 75 MHz) 63.28,55.01, 52.39, 49.20, 48.69, 47.00, 43.24, 42.77, 41.03, 40.27, 36.82,36.35, 35.75, 35.12, 32.77, 31.36, 30.10, 28.54, 27.88, 26, 96, 24.35,23.38, 18.18, 14.23, HRFAB-MS (thioglycerol+Na⁺ matrix) m/e; ([M+H]⁺)392.3627 (100%); calcd. 392.3641.

Octanyl cholate (328): Cholic acid (3.14 g, 7.43 mmol) and10-camphorsulfonic acid (0.52 g, 2.23 mmol) were dissolved in octanol(3.5 mL, 23.44 mmol). The solution was warmed to 40-50° C. in oil bathunder vacuum (˜13 mm/Hg). After 14 h, the remaining octanol wasevaporated under high vacuum. The crude product was purified viachromatography (silica gel, 5% MeOH in CH₂Cl₂) to afford the desiredproduct (2.81 g, 73% yield) as a white powder. ¹H NMR (CDCl₃, 500 MHz) δ4.06 (t, J=6.7 Hz, 2 H), 3.98 (s, 1 H), 3.86 (s, 1 H), 3.48-3.44 (m, 1H), 2.41-2.34 (m, 1 H), 2.28-2.18 (m, 3 H), 1.98-1.28 (series ofmultiplets, 35 H), 0.99 (d, J=3.3 Hz, 3 H), 0.90 (s, 3 H), 0.89 (t, J=7Hz, 3 H), 0.69 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 154.38, 73.18, 72.14,68.63, 56.07, 50.02, 49.32, 47.07, 46.74, 41.96, 41.67, 39.84, 39.76,35.66, 35.45, 34.95, 34.86, 34.15, 32.97, 32.91, 31.65, 31.11, 30.68,28.39, 27.78, 26.66, 26.52, 25.82, 25.70, 25.54, 25.15, 24.95, 23.45,22.69, 17.77, 12.71; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)543.4015 (100%), calcd. 543.4026.

Representative synthesis of compounds 329-331: Octanyl cholate (328)(0.266 g, 0.511 mmol), N-t-Boc-glycine (0.403 g, 2.298 mmol), DCC (0.474g, 2.298 mmol) and DMAP (0.0624 g, 0.051 mmol) were mixed in CH₂Cl₂ (15mL) for 3 h. The resulting white precipitate was removed by filtration.The filtrate was concentrated, and the product was purified bychromatography (silica gel, EtOAc/Hexane 1:2) to afford the desiredproduct (0.481 g, 95% yield) as a white powder. Compound 329 ¹H NMR(CDCl₃, 300 MHz) δ 5.18 (br, 3 H), 5.01 (s, 1 H), 4.61 (m, 1 H), 4.04(t, J=6.5 Hz, 2 H), 3.97-3.88 (series of multiplets, 6 H), 2.39-2.15(series of multiplets, 2 H), 2.06-1.02 (series of multiplets, 35 H),1.46 (s, 18 H), 1.45 (s, 9 H), 0.93 (s, 3 H), 0.88 (t, J=6.7 Hz, 3 H),0.81 (d, J=6 Hz, 3 H), 0.74 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 174.26,170.19, 169.9, 169.78, 155.87, 155.67, 79.95, 76.47, 75.167, 72.11,64.55, 47.40, 45.28, 43.17, 42.86, 40.82, 37.94, 34.71, 34.63, 34.43,31.86, 31.340, 31.20, 30.76, 29.29, 29.25, 28.80, 28.72, 28.42, 28.06,27.96, 27.19, 26.81, 26.29, 26.012, 25.66, 22.87, 22.71, 22.57, 17.55,14.18, 12.27; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺)-1014.6261 (100%), calcd. 1014.6242. Compound 330: ¹H NMR(CDCl₃, 500 MHz) δ 5.10 (s, 1 H), 4.92 (d, J=2.44 Hz, 1 H), 4.55 (m, 1H), 4.00 (t, J=6.8 Hz, 2 H), 3.39-3.33 (series of multiplets, 6 H),2.595-2.467 (series of multiplets, 6 H), 2.31-2.12 (series ofmultiplets, 2 H), 2.01-1.00 (series of multiplets, 37 H), 1.39 (s, 27H), 0.88 (s, 3 H), 0.84 (t, J=6.8 Hz, 3 H), 0.76 (d, J=6.3 Hz, 3 H),0.69 (s, 3 H); ¹³C NMR (CDCl₃, 75 MHz) δ 174.16, 172.10, 171.78, 171.67,155.95, 79.45, 75.67, 74.21, 71.10, 64.63, 47.79, 45.27, 43.52, 40.97,37.92, 36.35, 35.14, 35.05, 34.90, 34.71, 34.46, 31.91, 31.45, 30.95,29.35, 29.31, 28.96, 28.78, 28.56, 28.55, 27.22, 26.98, 26.269, 25.71,23.00, 22.77, 22.64, 17.75, 14.24, 12.39; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 1056.6702 (100%), calcd. 1056.6712. Compound 331¹³C NMR (CDCl₃, 125 MHz) δ 174.00, 172.75, 172.41, 172.30, 156.03,79.00, 75.28, 73.79, 70.77, 64.39, 47.43, 45.04, 43.21, 40.76, 40.00,39.93, 37.78, 34.74, 34.62, 34.23, 32.19, 32.01, 31.70, 31.24, 30.77,29.13, 29.10, 28.67, 38.58, 28.38, 25.86, 25.37, 22.56, 22.38, 17.51,14.05, 12.13; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)1098.7181 (100%), calcd. 1098.7181.

Representative synthesis of compounds 341-343: To compound 329 (0.463 g,0.467 mmol) was added HCl in dioxane (0.3 mL, 4.0 M). After stirring themixture for 30 min, the excess HCl and solvent were removed in vacuo.The product was isolated, after chromatography (silica gel,CH₂Cl₂/MeOH/NH₃.H₂O 10:1.2:0.1) as a (0.271 g, 84%) pale oil. Thetrihydrochloride salt of 341 was prepared by addition of HCl in dioxaneand evaporation of excess HCl and dioxane in vacuo giving a whitepowder. Compound 341: ¹H NMR (CDCl₃ with ˜10% CD₃OD, 500 MHz) δ 5.16 (s,1 H), 4.99 (t, J=3.6 Hz, 1 H), 4.61 (m, 1 H), 4.04 (t, J=6.8 Hz, 2 H),3.51-3.36 (m, 6 H), 2.34-2.15 (m, 2 H), 2.00-1.05 (series of multiplets,40 H), 0.93 (s, 3 H), 0.88 (t, J=7.1 Hz, 3H), 0.80 (d, J=3.2 Hz, 3 H),0.74 (s, 3 H); ¹³C NMR (CDCl₃ and ˜10% CD₃OD, 75 MHz) δ 174.32, 173.92,173.81, 76.08, 74.67, 71.61, 64.73, 47.64, 45.39, 44.41, 43.49, 40.97,37.99, 34.99, 34.77, 34.71, 34.52, 31.96, 31.54, 31.35, 30.96, 29.39,29.36, 29.02, 28.82, 27.32, 27.11, 26.11, 25.83, 23.01, 22.82, 22.69,17.79, 14.28, 12.41; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺)714.4651 (100%), calcd. 714.4669. Compound 342: ¹H NMR (CDCl₃ and ˜10%CD₃OD, 300 MHz) δ 5.142 (s, 1 H), 4.96 (d, J=2.7 Hz, 1 H), 4.60, (m, 1H), 4.04 (t, J=6.6 Hz, 2 H), 3.07-2.95 (series of multiplets, 6 H),2.56-2.43 (series of multiplets, 6 H), 2.38-2.13 (series of multiplets,2 H), 2.07-1.02 (series of multiplets, 36 H), 0.92 (s, 3 H), 0.88 (t,J-=6.6 Hz, 3 H), 0.82 (d, J=6.6 Hz, 3 H), 0.73 (s, 3 H); ¹³C NMR (CDCl₃and CD₃OD, 75 MHz) δ 174.29, 172.29, 171.98, 171.92, 75.52, 74.09,70.98, 64.67, 47.78, 45.26, 43.52, 40.98, 38.73, 38.62, 38.35, 38.07,38.03, 37.99, 35.01, 34.81, 34.77, 34.49, 31.92, 31.50, 31.40, 30.99,29.36, 29.33, 28.93, 28.80, 27.43, 26.96, 26.08, 25.56, 23.07, 22.79,22.62, 17.73, 14.25, 12.34; HRFAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 714.4651 (100%), calcd. 714.4669. Compound 343: ¹H NMR (CDCl₃and CD₃OD, 500 MHz) δ 5.12 (s, 1 H) 4.93 (s, 1 H), 4.59 (m, 1 H), 4.04(t, J=7 Hz, 2 H), 2.79-2.69 (series of multiplets, 6 H), 2.4621-2.2999(series of multiplets, 6 H), 2.2033-1.0854-(series of multiplets, 42 H),0.94 (s, 2 H), 0.91 (s, 1 H), 0.88 (t, J=7 Hz, 3 H), 0.82 (d, J=6.4 Hz,3 H), 0.75 (s, 3 H); ¹³C NMR (CDCl₃ and CD₃OD, 75 MHz) δ 174.70, 171.97,171.86, 171.75, 76.10, 74.55, 71.56, 64.85, 47.96, 45.31, 43.37, 40.87,38.09, 34.86, 34.80, 34.73, 34.46, 32.84, 32.62, 32.27, 31.87, 31.75,31.42, 31.08, 29.31, 29.28, 29.26, 28.78, 28.73, 27.38, 26.91, 26.05,25.37, 23.24, 23.15, 22.95, 22.74, 22.71, 22.43, 17.78, 14.11, 12.28;HRFAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 798.5624 (100%),calcd. 798.5609.

Benzyl cholate (312): Cholic acid (4.33 g, 10.62 mmol) and10-caphorsulfonic acid (0.493 g, 2.21 mmol) were dissolved in benzylalcohol (1.97 mL, 19.3 mmol). The suspension was heated to 50° C. in oilbath and stirred under vacuum (Q13 mm/Hg) for 16 h. Excess benzylalcohol was removed in vacuo, and the crude product was chromatographed(silica gel, 5% MeOH in CH₂Cl₂) to give the desired product as a whitepowder (4.23 g, 81% yield). ¹H NMR (CDCl₃, 500 MHz) 67.34-7.33 (m, 5 H),5.10 (d, J=1.5 Hz, 2 H), 3.92 (s, 1 H), 3.81 (s, 1 H), 3.42 (s, 1 H),3.40 (br, m, 3 H), 2.44-2.38 (m, 1 H), 2.31-2.25 (m, 1 H), 2.219 (t,J=12 Hz, 2 H), 0.96 (d, J=5.5 Hz, 3 H), 0.86 (s, 3 H), 0.63 (s, 3 H);¹³C NMR (CDCl₃, 125 MHz) δ 174.25, 136.30, 128.66, 128.63, 128.32,128.28, 128.24, 73.18, 71.98, 68.54, 66.18, 47.14, 46.56, 41.69, 39.65,35.51, 35.37, 34.91, 34.84, 31.49, 31.08, 30.50, 28.31, 27.62, 26.47,23.35, 22.65, 22.60, 17.42, 12.63, 12.57; HRFAB-MS (thioglycerol+Na⁺matrix) m/e: ([M+Na]⁺) 521.3235 (100%), calcd. 521.3242.

Representative synthesis of compounds 313-315: Benzyl cholate (312)(0.248 g, 0.499 mmol), N-t-Boc-glycine (0.404 g, 2.30 mmol), DCC (0.338g, 1.49 mmol) and DMAP (0.051 g, 0.399 mmol) were added to CH₂Cl₂ (15mL), and the suspension was stirred for 16 h. The resulting whiteprecipitate was removed by filtration, and the filtrate wasconcentrated. The product was obtained after chromatorgraphy (silicagel, EtOAc/Hexane 0.6:1) as a white powder (0.329 g, 68%). Compound 313:¹H NMR (CDCl₃, 300 MHz) δ 7.34-7.33 (m, 5 H), 5.16 (s, 1 H), 5.08 (dd,J=22.5 Hz, 12.3 Hz, 4 H), 5.00 (s, 1 H), 4.60 (m, 1 H), 4.04-3.81(series of multiplets, 6 H), 2.43-1.01 (series of multiplets, 25 H),1.46 (s, 9 H), 1.44 (s, 18 H), 0.92 (s, 3 H), 0.797 (d, J=5.7. Hz, 3 H),0.69 (s, 1 H); ¹³C NMR (CDCl₃, 75 MHz) δ 173.99, 170.25, 170.05, 169.85,155.73, 136.19, 128.69, 128.45, 128.35, 80.06, 77.65, 77.23, 76.80,76.53, 75.24, 72.19, 66.29, 47.46, 45.35, 43.24, 42.91, 40.89, 38.00,34.79, 34.66, 34.49, 31.43, 31.25, 30.77, 28.88, 28.40, 27.23, 26.89,25.74, 22.94, 22.65, 17.61, 12.32; FAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 992.5468 (100%), calcd. 992.5460.

Representative synthesis of compounds 316-318: Compound 313 (0.505 g,0.520 mmol) and Pd (5 wt. % on active carbon, 0.111 g, 0.0521 mmol) wereadded to MeOH (5 mL). The suspension was stirred under H₂ (50 psi) for20 hours. The solids were removed by filtration and the filtrate wasconcentrated. Purification of the product via chromatography (silicagel, 5% MeOH in CH₂Cl₂) gave a white powder (0.450 g, 98% yield).Compound 316: ¹H NMR (CDCl₃, 500 MHz) δ 5.20 (s, 1 H), 5.12 (br., 2H),4.92 (s, 1 H), 4.55 (m, 1 H), 3.98-3.83 (series of multiplets, 6 H),2.30-2.13 (series of multiplets, 2 H), 1.96-0.98 (series of multiplets,30 H), 1.40 (s, 9 H), 1.39 (s, 18 H), 0.87 (s, 3 H), 0.76 (d, J=6.3 Hz,3 H), 0.68 (s, 3 H); ¹³C NMR (CDCl₃ 75 MHz) δ 174.11, 165.60, 165.41,165.22, 151.28, 151.14, 75.48, 75.26, 71.81, 70.57, 67.50, 45.95, 42.58,40.65, 38.52, 38.16, 36.17, 33.28, 30.01, 29.78, 26.71, 26.42, 25.95,24.16, 23.78, 23.40, 23.31, 22.55, 22.16, 21.03, 18.23, 17.93, 12.91,7.61; FAB-MS (thioglycerol+Na⁺ matrix) m/e: ([M+Na]⁺) 902.4997 (21%),calcd. 902.4990.

Representative synthesis of compounds 319-321: Compound 316 (0.375 g,0.427 mmol), DCC (0.105 g, 0.512 mmol) and DMAP (0.062 g, 0.512 mmol)and N,N-dimethylethanolamine (0.09 ml, 0.896 mmol) were added to CH₂Cl₂(15 mL). The mixture for 16 h, and solvent and excessN,N-dimethylethanolamine were removed in vacuo. The product was purifiedvia chromatography (silica gel EtOAc/hexane/Et₃N, 12:10:0.6) giving awhite powder (0.330 g, 82% yield). ¹H NMR (CDCl₃ and ˜10% CD₃OD, 500MHz) δ 5.18 (s, 1 H), 5.00 (s, 1 H), 4.19 (t, J=5.0 Hz, 2 H), 3.92 (s, 3H), 3.81 (s, 3 H), 2.62 (t, J=10 Hz, 2 H), 2.30 (s, 6 H), 1.47 (s, 9H),1.47 (s, 1 H), 1.45 (s, 1 H), 2.12-1.05 (series of multiplets, 27 H),0.96 (s, 3 H), 0.84 (d, J=10.5 Hz, 3 H), 0.78 (s, 3 H); ¹³C NMR (CDCl₃and ˜10% CD₃OD, 125 MHz) δ 174.19, 170.05, 169.87, 156.21, 79.36, 79.27,76.06, 76.90, 71.80, 61.19, 57.04, 46.88, 44.87, 44.67, 44.53, 42.78,42.15, 42.01, 40.43, 37.47, 34.32, 34.11, 33.92, 33.35, 33.25, 30.74,30.56, 30.16, 28.40, 27.67, 27.62, 26.73, 26.19, 25.18, 25.10, 24.72,24.49, 22.29, 21.81, 16.76, 11.56; FAB-MS (thioglycerol+Na⁺ matrix) m/e:([M+Na]⁺) 973.5723 (100%), calcd. 973.5725. The white solid from theprevious reaction (0.680 g, 0.714 mmol) and MeI (1 M in CH₂Cl₂, 1.5 mL)were stirred together for 2 h. The solvent and excess MeI were removedin vacuo giving a white solid (0.812 g 100%). The product was carried onwithout further purification.

Representative synthesis of compounds 324-326: Compound 319 (0.812 g,0.714 mmol) was dissolved in CH₂Cl₂ (5 mL) and trifluoroacetic acid (0.5mL) was added. The mixture was stirred for 16 min. The solvent andexcess acid were removed in vacuo, and the resulting oil waschromatographed (silica gel, CH₂Cl₂/MeOH/NH₃—H₂O 4:4:1) to give thedesired product as a pale glass (0.437 g, 90% yield). Addition of HCl (2M in ethyl ether, 2.5 mL) gave the trihydrochloride salt of 324 as apale yellow powder. Compound 324: ¹H NMR (50% CDCl₃, 50% CD₃OD, 300 MHz)δ 5.43 (s, 1H), 5.24 (s, 1 H), 4.84 (m, 1 H), 4.66 (m, 2 H), 4.16-3.96(series of multiplets, 6 H), 3.88 (m, 2 H), 3.37 (s, 9 H), 0.67 (s, 3H), 0.59 (d, J=6.3 Hz, 3 H), 0.56 (s, 3 H); ¹³C NMR (50% CDCl₃, 50%CD₃OD, 75 MHz) δ 173.47, 167.06, 167.01, 166.70, 78.01, 76.49, 73.78,64.98, 57.67, 53.36, 47.49, 46.99, 45.61, 43.28, 40.83, 40.23, 40.10,37.69, 34.80, 34.48, 34.28, 31.03, 30.63, 30.44, 28.94, 27.05, 26.56,25.50, 22.53, 21.56, 16.95, 11.37; FAB-MS (thioglycerol+Na⁺ matrix) m/e:([M−I]⁺) 665.4475 (85;6%), cacld 665.4489. Compounds 325 and 326 provedtoo unstable to chromatograph using the basic eluent used for thepurification of 324. Consequently, 325 and 326 were prepared bydeprotection of 320 and 321 using HCl (2 M in diethyl ether), followedby tituration with ethyl acetate. The compounds were then used withoutfurther purification. ¹H NMR spectroscopy indicated that compounds 325and 326 were >95% pure. Compound 325: ¹H NMR (50% CDCl₃, 50% CD₃OD, 500MHz) δ 5.21 (s, 1 H), 5.02 (d, J=4 Hz, 1 H), 4.64 (m, 1 H), 4.53 (m, 2H), 3.74 (m, 2 H), 3.31-3.01 (series of multiplets, 6 H), 3.23 (s, 9 H),2.96-2.73 (series of multiples, 6 H), 2.51-2.44 (m, 1 H), 2.35-2.29 (m,1 H), 2.14-1.09 (series of multiplets, 26 H), 0.99 (s, 3 H), 0.85 (d,J=6.5 Hz, 3 H), 0.80 (s, 3 H); ¹³C NMR (50% CDCl₃, 50% CD₃OD, 125 MHz) δ172.77, 169.88, 169.56, 169.50, 75.94, 74.44, 71.57, 64.31, 56.94,52.92, 46.78, 44.59, 42.70, 40.21, 37.16, 34.80, 34.72, 34.66, 34.05,34.00, 33.78, 33.62, 30.95, 30.91, 30.81, 30.41, 29.96, 29.81, 28.20,26.37, 26.06, 24.74, 24.24, 22.04, 21.13, 16.54, 10.97; FAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M−I]⁺) 707.4958 (25.6%), cacld707.4958. Compound 326: ¹H NMR (50% CDCl₃, 50% CD₃OD, 500 MHz) δ 5.12(s, 1 H), 4.94 (d, J=2.5 Hz, 1 H), 4.56 (m. 1 H), 4.51 (t, J=2.3 Hz, 2H), 3.74 (m, 2 H), 3.23 (s, 9 H), 3.05-3.01 (m, 4 H), 2.98 (t, J=7.5 Hz,2 H), 2.63-2.43 (series of multiplets, 6 H), 2.31-2.24 (series ofmultiplets, 2 H), 2.07-1.87 (series of multiplets, 12 H), 1.17-1.05(series of multiplets, 23 H), 0.94 (s, 3 H), 0.82 (d, J=6.0 Hz, 3 H),0.76 (s, 3 H); ¹³C NMR (50% CDCl₃, 50% CD₃OD, 125 MHz) δ 171.87, 169.79,169.59, 169.50, 76.12, 74.70, 71.65, 65.57, 65.08, 64.40, 57.68, 53.74,52.78, 45.33 43.54, 41.04, 39.12, 37.92, 43.85, 34.72, 34.56, 34.34,32.30, 31.47, 31.27, 30.87, 30.58, 29.03, 27.053, 26.84, 25.51, 24.95,24.91, 22.87, 22.82, 22.65, 21.93, 17.31, 11.81; FAB-MS(thioglycerol+Na⁺ matrix) m/e: ([M−I]⁺) 749.5432 (100%), cacld 749.5436.

Example 14 Stability Tests of Compounds 352-354

Compounds 352-354 were dissolved in 50 mM phosphate buffered water (pH2.0, 7.0 or 12.0) at approximately 10 mM concentrations. The structuresof compounds 352-354 are given in FIG. 9. Decomposition of the compoundswas observed via HPLC (cyano-silica column, 0.15% TFA water-acetonitrilegradient elution). Table 15 shows the stabilities (half-lives) ofcompounds 352-354 in phosphate buffer at room temperature, pH 2.0, pH7.0 and pH 12.0. These compounds were used since they contain achromophore that facilitated monitoring of decomposition by absorptionmethods common in the HPLC apparatus used.

At low pH, the amines are expected to be protonated and the compoundsshowed relative stability. At higher pH, the amines were less stronglyprotonated and became involved in ester hydrolysis. The γ-aminobutyricacid-derived compound was especially susceptible to hydrolysis,presumably yielding pyrrolidone. In general, the compounds are believedto hydrolyse to give cholic acid, choline or octanol, and glycine,beta-alanine, or pyrrolidone, depending on the particular compound.

Decomposition through ester hydrolysis yielded compounds that were lesspolar and easily separable from the starting compounds. Initially, onlyone benezene-containing decomposition product was observed; at longerreaction times, two other decomposition products were observed whichpresumably corresponded to sequential ester hydrolysis.

Example 15 Further Syntheses of Compounds of Formula I

Compounds of formula I can also be prepared as shown in the followingscheme.

Alterations in the stereochemistry within the steroid (AB ring juncturein this case)

Alterations in the saturation within the steroid (AB ring juncture inthis case)

Alterations in the number of hydroxyl groups on the steroid (OH-12 inthis case)

Alterations in other groups on the steroid (in the A ring in this case)

Descriptions of the steroid starting materials shown above can be foundin Dictionary of Steroids, Hill, R. A.; Kirk, D. N.; Makin, H. L. J.;Murphy, G. M., eds., Chapman and Hall: New York, 1991.

Example 15 Testing of Compounds With Gram-Negative Bactenra MIC and MBCMeasurements

General: Microorganisms. Reference strains were purchased from theAmerican Type Culture Collection (Rockville, Md.) or Bactrol disks fromDifeo Laboratories (Detroit, Mich.). The following specific ATCC strainswere used: 10798 Escherichia coli, 25922 Escherichia coli, 13883Klebsiella pneumoniae, 27853 Pseudomonas aeruginosa, 14028 Salmonellatyphimurium, 29212 Enterococcus faecalis, 25923 Staphylococcus aureus,19615 Streptococcus pyogenes, and 90028 Candida albicans. Bacterialstrains were maintained on Mueller-Hinton agar plates, and Candidaalbicans was maintained on Sabouraud Dextrose agar plates.

Tryptic soy broth (TSB) was made by dissolving 27.5 grams of tryptic soybroth without dextrose (DIFCO Laboratories) in 1 liter of deionizedwater and sterilizing at 121° C. for 15 minutes. Solid agar (TSA) plateswere made by dissolving 6.4 grams of tryptic soy broth and 12 grams ofagar (purified grade, Fischer Scientific) in 800 mL of deionized waterand sterilizing at 121° C. for 20 minutes. Aliquots (20 mL) of thehomogeneous solution were then poured in sterile plastic petri dishes(100×15 mm, Fisher Scientific). Solutions of compounds were made bydissolving the HCl salt of the respective compound into an appropriateamount of deionized and sterilized water followed by microfiltration.

Representative procedure for measuring MIC and MBC values: A suspensionwas prepared of E. coli (ATCC 10798) containing ˜10⁶ CFU (colony formingunits)/mL from a culture incubated in TSB at 37° C. for 24 hours.,Aliquots of 1 mL of the suspension were added to test tubes containing 1mL TSB and incrementally varied concentrations of cholic acidderivatives and/or erythromycin or novobiocin. In the sensitizationexperiments, erythromycin or novobiocin were added 15 minutes later thanthe cholic acid derivatives. The samples were subjected to stationaryincubation at 37° C. for 24 hours. Sample turbidity was determined bymeasuring absorption at 760 nm (HP 8453 UV-Visible Chemstation, HewlettPackard). Additionally, an alliquot from each of the samples showing nomeasurable turbidity was subcultured on TSA plates (alliquots werediluted to provide fewer than 300 CFU). Colonies that grew on thesubculture after overnight incubation were counted and the number ofCFU/mL in the samples were calculated. The calculated values werecompared to the number of CFU/mL in the original inoculum. MIC valueswere determined as the concentrations of the studied compounds at whichthe number of CFU/mL remained constant or decreased after incubation for24 hours. The MBC values were determined as the lowest concentrations ofthe studied compounds that allowed less than 0.1% of the originalbacterial suspension to survive.

Example 16 Demonstration of Membrane Disrupting Properties of the CholicAcid Derivatives

Using a technique described by J. M. Shupp, S. E. Travis, L. B. Price,R. F. Shand, P. Keim, RAPID BACTERIAL PERMEABILIZATION REAGENT USEFULFOR ENZYME ASSAYS, Biotechniques, 1995, vol. 19, 18-20, we have shownthat the cholic acid derivatives increase the permeability of the outermembrane of Gram-negative bacteria. The values for half maximumluminescence (indicating permeabilization of the outer membrane allowingluciferin to enter the cell) for 2 is 7 μg/mL and for 10 is 33 μg/mL.These values correspond to the measured MICs of 2 and 10.

PMB is known to have membrane permeabilization and bactericidalproperties. PMB has a hydrophobic acyl group and a macrocylicheptapeptide containing a D amino acid and four diaminobutyric acid(DAB) residues. One of the DAB side chains is involved in forming themacrocylic ring, leaving the other three side chains with free amines.Thus, PMB has an array of amines oriented on one face, or plane, of ahydrophobic scaffolding. It has been suggested that the primary role ofthe macrocylic ring is to orient the amine groups in a specificarrangement necessary for binding the lipid A portion of LPS. Therelative spatial orientation of these primary amine groups is the samein the cholic acid derivatives as in PMB.

The stereochemistry of the steroid backbone results in differentactivities of the cholic acid derivatives (compare 2 and 8, Tables 1, 2,6 and 7). Compounds with guanidine groups attached to the steroid havelower MIC values than compounds containing amine groups (compare 1, 2, 4and 5, compare Tables 1-8). The length of the tether between the amineor guanidine groups and the steroid backbone also influences activity(compare 1-3, Tables 1, 2, 6 and 7). Ester tethers between amine groupsand the steroid backbone provide compounds with MIC values that arehigher than the corresponding compounds containing ether tethers(compare 1, 2, 6 and 7, tables 1 and 2).

The group attached to the backbone at C-20 or C-24 also influences theactivity of the cholic acid derivatives. A long carbon chain attached tothe steroid via an ether linkage at C-24 lowers the MIC of the compoundas compared to the compound with a hydroxyl group at C-24 (compare 2, 9and 10, Tables 1, 2, 6 and 7). Short chains of carbon or oxygen attachedat C-20 decrease the MIC values of the cholic acid derivatives (compare10 and 11, Tables 1 and 2). Covalently linking the cholic acidderivatives increases the activity of the compounds (compare 10 and 12,Tables 1 and 2).

Ability to permeabilize outer membrane: Compounds 11, 106, and 108-114(FIG. 1) were tested for antibiotic activity. They were also tested forthe ability to permeabilize the outer membrane of Gram-negativebacteria, causing sensitization to hydrophobic antibiotics that cannotcross the outer membrane. The permeabilization of the outer membrane wasmeasured using erythromycin and novobiocin. These antibiotics are activeagainst Gram-positive bacteria, but inactive against Gram-negativebacteria, due to the barrier formed by the outer membrane ofGram-negative bacteria.

Most of the experiments were performed with Escherichia coli K-12 strainATCC 10798; however, to demonstrate that the activity of the cholic acidderivatives was not species dependent, the activity of selectedcompounds was also measured with Pseudomonas aeruginosa (ATCC 27853).The MICs of erythromycin and novobiocin against E. coli (ATCC 10798) at70 and >500 μg/mL were measured. The threshold measure ofpermeabilization was the concentration of the cholic acid derivativesrequired to lower the MIC of either erythromycin or novobiocin to 1μg/mL.

Results of the MIC, MBC and permeabilization (with erythromycin)measurements are shown in FIG. 2 (in FIG. 2, Compound A is polymyxin Bnonapeptide). As FIG. 2 illustrates, the MIC and MBC values of thecompounds dropped dramatically as the length of the side chain extendingfrom C-17 increased. The apparent role of the hydrophobic steroid sidechain is to facilitate membrane insertion and self-promoted transportafter initial association with the outer membrane of Gram-negativebacteria (as shown in FIG. 3). Outer membrane permeabilization occurs asa result of association with the lipid A on the outer leaflet of themembrane. Permeabilization of the outer membrane alone does not causecell death, suggesting that the compounds must pass through the outermembrane to kill bacteria. This ability to traverse the outer membrane,and thereby disrupt the cytoplasmic membrane, is required for thecompounds to have lethal activity.

As observed, compounds lacking a hydrophobic side chain are lesseffective in killing bacteria. It is hypothesized that these compoundsare capable of permeabilizing the outer membrane (i.e., associating withthe lipid A on the outer leaflet of the membrane), but incapable ofcrossing through the outer membrane.

The fractional inhibition concentration (FIC) values of the compounds,were calculated using erythromycin and novobiocin as the secondarycompounds. With the exception of 114, the compounds displayed FIC valuesof less than 0.5 with erythromycin, with some values near 0.05 (Table9).

Details from studies with novobiocin are also shown in Table 9. The factthat results with erythromycin and novobiocin were comparabledemonstrates that the activity of the cholic acid derivatives is notantibiotic-dependent. Similar trends were observed with E. coli (ATCC10798) and P. aeruginosa (ATCC 27853), although, as expected, P.aeruginosa was more resistant than E. coli. These results suggest thatthe activity of the compounds tested is not species-dependent.

Compounds with hydrophobic alkylaminoalkyl side chains were prepared(compounds 133 and 134, FIG. 4). As observed with other compounds, theincorporation of guanidine groups (in 134) increased the activity of thecholic acid derivatives as compared to compounds containing primaryamines. As a control, 135 (FIG. 4), which did not have a hydrophobicside chain, was prepared. The MIC of the control (135) was relativelyhigh, as expected, as was the MBC (FIG. 5). In contrast, the MICs of 133and 134 were very low; in fact they rivaled PMB in activity. Notably,the MBCs of 133, 134, and PMB were very similar to the MICs; that is, ata threshold concentration these compounds killed all of the bacteria insolution.

As an additional means of demonstrating the membrane disruptingcapabilities of the cholic acid derivatives 133 and 134, aluciferin/luciferase-based cell lysis assay was used (as described inWillardson et al., Appl. Environ. Microbiol. 1998, 64, 1006 and Schuppet al., Biotechniques 1995, 19, 18). In this assay, E. coli containingan inducible luciferase coding plasmid was incubated with the inducingagent (toluene), then treated with a lysis buffer containing either PMBor one of the cholic acid derivatives, and Triton X-100. Luciferin andATP were then added. Cell lysis resulted in luminescence. Theconcentrations of the membrane disrupting agents (PMB and the cholicacid derivatives) were varied, and the resulting luminescence wasmeasured. In the absence of the membrane disrupting agents, noluminescence was observed.

The MICs of 133, 134 and. PMB and the concentrations required for halfmaximal luminescence are shown in FIG. 6. As is the case with the MICvalues, the compounds 133 and 134 rival PMB in activity in theluminescence assay.

Effect of sulfate group: To observe if the presence of a sulfate groupat C-24 in a cholic acid derivative would increase the activity of thecompounds, 132 (shown in FIG. 7) was tested. The MIC of 132 with E. coli(ATCC 10798) was 60 μg/mL. The concentration required to lower the MICof erythromycin to 1 μg/mL was 4.0 μg/mL with the same strain. Theantibiotic and permeabilization activities of 132 were lower than thoseof the parent alcohol 110 (shown in FIG. 1).

Additional experiments: Additional experiments were carried out usingcompounds 1, 2, 5, 106, 10, 112, 133, and 134. MIC and MBC data forthese compounds with representative strains of Gram-negative andGram-positive organisms are shown in Table 10. For comparison purposes,the MICs of PMB with various organisms were also measured and arepresented in Table 10.

In addition to PMB, compounds 1, 2, 5, 106, 10, 112, 133, and 134 sharesome features with other steroid antibiotics. For example, squalamineincludes a steroid nucleus and a polyamine side chain (Moore et al.,Proc. Natl. Acad. Sci. 1993, vol. 90, 1354-1358). It is proposed thatsqualamine incorporates into lipid bilayers and thus disrupts thebacterial membrane. In squalamine, the polar polyamine functionality islocated at the distal end of the molecule, leaving a hydrophobic core.In 1, 2, 5, 106, 10, 112, 133, and 134, the amines are located on oneside of the steroid, giving compounds that are facially anphiphilic. Anadditional series of compounds related to 1, 2, 5, 106, 10, 112, 133,and 134 includes cholic acid derivatives with amines at C-24 (e.g., 140in FIG. 7). In contrast to 1, 2, 5, 106, 10, 112, 133, and 134, thesecompounds have been shown to have only weak antibacterial activityagainst Gram-positive strains and no activity against Gram-negativestrains.

The cholic acid derivatives 1, 2, 5, 106, 10, 112, 133, and 134 displaya range of activities, some with submicrogram per milliliter MICs. Withmany organisms, MIC and MBC values are very similar, especially with themost active compounds. Some of the compounds have lethal activity,presumably due to disruption of the cytoplasmic membrane. Others haveonly sublethal activity, due to permeabilization of the outer membrane.

Compounds lacking a hydrophobic chain (e.g., 106 and 10) have high MICvalues, but are effective permeabilizers of the outer membrane ofGram-negative bacteria. Because these compounds lack a hydrophobicchain, they have sublethal, but not lethal activity against thesebacteria. Compounds with hydrophobic chains (e.g., 133 and 134) havelethal activity.

The hemolytic behavior of the cholic acid derivatives 1, 2, 5, 106, 10,112, 133, and 134 suggests that they can act as membrane-disruptingagents, and their antimicrobial activity likely involves membranedisruption. With Gram-negative strains, the target of inactivity isexpected to be the cytoplasmic membrane., Compounds such as 106 and 10ineffectively cross the outer membrane and do not display lethalactivity. The hydrophobic chains in 133 and 134 may facilitateself-promoted transport across the outer membrane, allowing them todisrupt the cytoplasmic membrane.

The results shown in Table 10 indicate that the presence of ahydrophobic chain is much less important for lethal activity againstGram-positive strains. Without the requirement for crossing an outermembrane, compounds lacking a hydrophobic chain extending from C-17 caneffectively kill Gram-positive bacteria.

Various tether lengths were investigated to determine the optimalspacing of the amine or guanidine groups from the steroid. It was foundthat three carbon tethers gave compounds that were more effective thanthose with two carbon tethers (e.g., compare the MICs of 1 with those of2. The resultant increase in antibiotic activity upon substitution ofguanidine groups for amines suggests a central role foramine/guanidine-phosphate interactions.

The nature of the group attached to the steroid backbone at C-17 greatlyinfluenced the activity of the compounds with Gram-negative bacteria.For example, the differences among the MIC and MBC values for 106, 10,and 112 were notable. This trend was also observed in the MIC and MCBvalues of 2 and 5, as compared to those of 133 and 134 (in thiscomparison, the benzyl groups in 2 and 5 are expected to be lesshydrophobic than the octyl chains found in 133 and 134). The influenceof the group attached to the steroid at C-17 is less pronounced withGram-positive strains; e.g., 5 and 134 have similar MIC values withStaphylococcus aureus.

To measure permeabilization, the FIC values for compounds 1, 2, 5, 106,10, 112, 133, and 134 with erythromycin, novobiocin, and rifampicin weredetermined. Concentrations of 0.5, 1.0 or 3.0 μg/mL of these antibioticswere used, and the concentrations of the cholic acid derivativesrequired to inhibit bacterial growth of Gram-negative strains weredetermined. The concentrations required for bacterial growth inhibitionand the FIC values are shown in Tables 11-13. Interestingly, the MICvalues of the compounds do not directly correlate with their ability topermeabilize the outer membrane. For example, compounds 106 and 10 haverelatively high MIC values, but are potent permeabilizers. Nearly all ofthe compounds demonstrated FIC values of less than 0.5, with some lessthan 0.03. The cholic acid derivatives that give relatively high FICvalues (i.e., 5, 133, and 134) are themselves potent antibiotics.

Ester and amide side chains: Additional compounds, for example,compounds with amide and ester side chains, were tested. Compounds 203b,6, and 210a (Scheme 12) displayed potent synergism with erythromycin andnovobiocin (Table 14). In the triester series (203a, 203b, 6, and 7),the β-alanine derived compounds are more active than those derived fromglycine. Substitution at C24 had minimal effect on the activity of thesecompounds (compare 203b and 7).

Triamides 209a-c (Scheme 12) were less active than the esters, possiblydue to conformational constraints imposed by the amide bonds. With thetriamides, substitution at C24 had significant effects on the activityof the compounds (compare 209a and 210a, Table 14). In this series, theglycine derivative was more active than the corresponding β-alaninederivative.

The relative lack of synergism displayed by the lysine derivative may beattributable to the length of the side chain. As a control, compound 211(FIG. 8), a derivative of 209c lacking the α-amino group, was prepared;this compound was less active than 209c as a permeabilizer. Compound 206also proved to be ineffective as a permeabilizer. These results suggestthat the optimal length for the tether between the steroid and the aminefunctionality is between zero and six atoms.

Further compounds 341-343 and 324-327 were, investigated similarly. Thestructures of compounds 341-343 and 324-327 are shown in FIG. 11. TheMIC of these compounds against S. aureus (ATTC 25923) are presented inTable 16 as MIC^(a), the MIC of these compounds against E. coli (ATTC25922) are presented in Table 16 as MIC^(b), the concentration of thecompounds required to lower the MIC of erythromycin from 36 to 1 μg/mLwith E. coli is shown in Table 16 as c, and the minimum hemolyticconcentration of the compounds is shown in Table 16 as MHC^(d). Minimuminhibition concentrations (MIC) and minimum hemolytic concentrations(MHC) were measured as described in Li et al., Antimicrob. Agents.Chemother., 1999, 43, 1347. The compounds in Table 16 containing ahydrophobic chain at carbon 24 (341-343) were the most active againstthe Gram-negative strain. The compounds with choline at carbon 24(324-326) were much less active alone against the Gram-negative strain,yet retained the ability to inhibit the growth of the Gram-positivestrain. This difference may be due to the inability of compounds 324-326to traverse the ourter membrane of the Gram-negative strain studiedhere. Compound 327 was inactive. Compounds 341-343 exhibited very lowMHC; however, compounds 324-326 appear to be non-hemolytic, presumablydue to the additional positive charge at carbon 24. The structures ofcompounds 356-358 are shown in FIG. 12. An outline of the synthesis ofcompounds 356-357 is shown in Scheme 16. TABLE 1 Measurement of MIC andMBC values of 1-12 with E. coli (ATCC 10798) Compound MIC (μg/mL) MBC(μg/mL) 1 20 34 2 7 16 3 6 a 4 5 10 5 2  4 6 65 a 7 28 a 8 46 a 9 3 1010 36 60 11 140 >160  12 4  4^(a)Value not measured.

TABLE 2 Measurement of the concentrations of 1-12 required to lower theMIC of erythromycin from 70 μg/mL to 1 μg/mL with E. coli (ATCC 10798).Compound MIC (μg/mL) MBC (μg/mL) 1 2 20 2 1 10 3 1.5 a 4 1.5 10 5 1 3 622 a 7 2.5 a 8 10 a 9 3 3 10 2 50 11 40 >160 12 1.5 2.5^(a)Value not measured.

TABLE 3 Measurement of the concentrations of 1, 2, 4 and 5 required tolower the MIC of novobiocin from >500 μg/mL to 1 μg/mL with E. coli(ATCC 10798). Compound MIC (μg/mL) MBC (μg/mL) 1 20 34 2 7 16 4 5 10 5 24 11 40 140 12 2.5 a^(a)Value not measured.

TABLE 4 Measurement of MIC and MBC values of 1, 2, 4 and 5 with E. coli(ATCC 25922). Compound MIC (μg/mL) MBC (μg/mL) 1 25 40 2 10 20 4 6 9 5 24

TABLE 5 Measurement of the concentrations of 1, 2, 4 and 5 required tolower the MIC of erythromycin from 60 μg/mL to 1 μg/mL with E. coli(ATCC 25922). Compound MIC (μg/mL) MBC (μg/mL) 1 2 14 2 1 5 4 1 5 5 1.51.5

TABLE 6 Measurement of MIC and MBC values of 1-5, 8-12 with P.aureginosa (ATCC 27853). Compound MIC (μg/mL) MBC (μg/mL) 1 15 >50  2 940 3 16 a 4 15 40 5 6 15 8 50 a 9 8 a 10 23 a^(a)Value not measured.

TABLE 7 Measurement of the concentrations of 1-5, 8-12 required to lowerthe MIC of erythromycin from 240 μg/mL to 5 μg/mL with P. aureginosa(ATCC 27853). Compound MIC (μg/mL) MBC (μg/mL) 1 8 45 2 4 25 3 6 a 4 540 5 3 10 8 40 a 9 5 a 10 7 a^(a)Value not measured.

TABLE 8 Measurement of the concentrations of 1, 2, 4 and 5 required tolower the MIC of novobiocin from >500 μg/mL to 1 μg/mL with P.aureginosa (ATCC 27853). Compound MIC (μg/mL) 1 6 2 4 4 6 5 6

TABLE 9 Com- MIC MBC (a) (b) (d) pound (μg/mL) (μg/mL) (μg/mL) (μg/mL)FIC^(c) (μg/mL) FIC^(e) 106 140 >200 30 160 0.23 50 0.36 11 140 >160 20180 0.16 40 0.29 108 70 140 4.0 140 0.071 12 0.17 109 70 120 4.0 800.071 15 0.22 110 36 60 2.0 50 0.070 4.0 0.11 111 30 33 1.0 20 0.048 2.00.069 112 12 17 0.4 4.0 0.048 0.8 0.085 113 3.0 5.0 0.8 2.0 0.28 1.00.27 114 3.0 10 3.0 3.0 1.0 n.d. n.d.MIC, MBC, permeabilization and FIC data with Escherichia coli (ATCC10798).(a) Concentration required to lower the MIC of erythromycin from 70 to 1μg/mL.(b) MBC with 1 μg/mL erythromycin.^(c)FIC values with erythromycin.(d) Concentration required to lower the MIC of novobiocin from >500 to 1μg/mL.^(e)FIC values with novobiocin.

TABLE 10 1 (μg/mL) 2 (μg/mL) 5 (μg/mL) 106 (μg/mL) 10 (μg/mL) 112(μg/mL) 133 (μg/mL) 134 (μg/mL) PMB (1) ORGANISM MIC MBC MIC MBC MIC MBCMIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC Gram-negative rodsEscherichia 22 22 5.1 6.8 1.4 3.8 80 90 36 40 6.6 7.4 3.0 3.0 0.31 0.371.8 1.8 coli ATCC 25922 Klebsiella 24 >36 14 17 3.0 6.7 >100 100 47 5023 27 2.6 5.8 0.84 3.0 5.3 6.8 pneumonia ATCC 13883 Pseudomonas 26 3811 >17 5.9 9.9 85 97 21 36 4.6 6.4 2.0 3.2 2.0 2.9 0.20 3.9 aeruginosaATCC 27853 Salmonella 21 >25 13 16 2.2 3.8 >100 >100 43 >17 86 90 2.66.7 0.81 1.8 nm nm typhimurium ATCC 14028 Gram-positive cocclEnterococcus 4.9 50 3.4 19 2.2 16 12 >100 3.3 19 3.1 4.7 3.1 5.5 3.0 5.840 >100 faecalis ATCC 29212 Staphylococcus 3.1 5.7 1.0 4.7 0.6 3.2 8.654 2.0 9.2 0.55 4.2 0.4 2.0 0.59 1.4 26 >100 aureus ATCC 25923Streptococcus 3.0 4.4 2.0 2.3 2.0 2.1 18 37 4.2 5.8 2.4 3.0 2.3 2.9 3.53.5 9.0 16.3 pyrogenes ATCC 19615 Candida 49 >50 30 42 11 50 75 92 14 2941 45 31 45 53 76 albicans ATCC 90028 MHC 78 58 26 >100 100 5.9 29 9.0

TABLE 11 1 2 5 106 10 112 133 134 μg/ μg/ μg/ μg/ μg/ μg/ μg/ μg/ORGANISM a mL FIC mL FIC mL FIC mL FIC mL FIC mL FIC mL FIC ml FICEscherichia coli 30 2.5 0.15 0.23 0.078 0.38 0.31 3.2 0.073 1.5 0.0740.59 0.12 1.2 0.42 0.37 0.36 ATCC 25922 Klebsiella 33 1.0 0.072 0.250.048 0.15 0.080 3.6 0.66 0.21 0.035 0.1 0.035 0.11 0.073 0.18 0.24pneumonia ATCC 13883 Pseudomonas >100 6.6 0.29 2.4 0.25 2.3 0.42 16 0.222.1 0.13 1.0 0.59 0.35 0.21 0.70 0.42 aeruginosa ATCC 27853 Salmonella61 3.6 0.19 2.0 0.17 0.46 0.23 7.1 0.088 0.87 0.037 0.20 0.019 0.72 0.290.34 0.44 typhimurium ATCC 14028

TABLE 12 1 2 106 10 112 ORGANISM a μg/mL FIC μg/mL IC μg/mL FIC μg/mLFIC μg/mL FIC Escherichia coli 41 0.35 0.041 0.33 0.089 4.7 0.084 0.300.033 0.40 0.085 ATCC 25922 Klebsiella pneumonia 75 4.7 0.21 0.49 0.0488.9 0.10 0.73 0.029 0.19 0.022 ATCC 13883 Pseudomonas aeruginosa >1003.9 0.16 2.9 0.27 30 0.36 5.3 0.26 0.72 0.17 ATCC 27853 Salmonellatyphimurium >100 4.4 0.22 4.5 0.36 8.4 0.094 1.8 0.052 0.39 0.015 ATCC14028

TABLE 13 1 2 106 10 112 ORGANISM a μg/mL FIC μg/mL IC μg/mL FIC μg/mLFIC μg/mL FIC Escherichia coli 7.6 0.74 0.099 0.80 0.22 4.2 0.12 0.700.085 0.81 0.19 ATCC 25922 Klebsiella pneumonia 19 0.40 0.043 0.12 0.0351.8 0.044 0.16 0.030 0.11 0.031 ATCC 13883 Pseudomonas aeruginosa 26 1.50.096 0.50 0.086 11 0.17 0.84 0.083 0.50 0.15 ATCC 27853^(b) Salmonellatyphimurium 21 0.84 0.089 0.39 0.079 1.4 0.064 0.55 0.063 0.10 0.051ATCC 14028^(b)

TABLE 14 MIC Compound (μg/mL) a (μg/mL) b (μg/mL) 203a 85 18 55 203b 804 10  6 85 15 40  7 70 3 13 209a >100 25 75 209b >100 40 75 209c 85 4560 210a 80 6 18 210b 100 15 40a: concentration of cholic acid derivatives required to lower MIC oferythromycin to 1 μg/ML.b: concentration of cholic acid derivatives required to lower MIC ofnovobiocin to 1 μg/ML.

TABLE 15 Compound pH 2.0 pH 7.0 pH 12.0 352 >37 days 28 days <30 minutes353 >37 days 37 days <30 minutes 354  33 days 12 days <30 minutes

TABLE 16 MIC MIC MHC Compound (μg/mL) (μg/mL) c (μg/mL) (μg/mL) 341 1.81.0 0.7 4.0 342 4.0 7.0 3.0 2.0 343 1.2 3.5 3.5 <10 324 15 60 10 >200325 11 30 2.0 >200 326 14 23 2.0 >200 327 >100 >100 >100 >100

Other Embodiments

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. For examples, salts, esters, ethers and amides of novelsteroid compounds disclosed herein are within the scope of thisinvention. Thus, other embodiments are also within the claims.

1. A compound according to formula I

wherein: fused rings A, B, C, and D are independently saturated or fullyor partially unsaturated; and R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆,and R₁₇ is each independently selected from the group consisting ofhydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,(C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10)alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl,(C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl, C2-C6alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, asubstituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, asubstituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substitutedor unsubstituted (C1-C10) aminoalkylaminocarbonyl, a substituted orunsubstituted (C1-C10) aminoalkylcarboxamido, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyl oxy, (C1-C10)quaternaryammoniumalkylcarboxy, and (C1-C10) guanidinoalkyl carboxy,where Q5 is a side chain of any amino acid, P.G. is an amino protectinggroup, and R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is each independently: deletedwhen one of fused rings A, B, C, or D is unsaturated so as to completethe valency of the carbon atom at that site, or selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, alinking group attached to a second steroid, a substituted orunsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted(C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)—C(O)—O—,(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5is a side chain of any amino acid, P.G. is an amino protecting group,and provided that at least two of R₁ through R₁₄ are independentlyselected from the group consisting of a substituted or unsubstituted(C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl,(C1-C10) quaternaryammonium alkylcarboxy, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyloxy, and (C1-C10)guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof. 2.The compound of claim 1, wherein at least one of the following pairs isdeleted and the valency of the ring carbon atoms at these deletedpositions is completed with a double bond: R₅, and R₉; R₈ and R₁₀; andR₁₃ and R₁₄.
 3. The compound of claim 1, wherein at least three of R₁through R₁₄ are independently selected from the group consisting of asubstituted or unsubstituted (C1-C10) aminoalkyloxy, (C1-C10)alkylcarboxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, (C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10)alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted orunsubstituted (C1-C10) aminoalkyl, a substituted or unsubstituted(C1-C10) aminoalkylaminocarbonyl, a substituted or unsubstituted(C1-C10) aminoalkylcarboxamido, a substituted or unsubstitutedarylamino-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkyloxy-(C1-C10) alkyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, (C1-C10)quaternaryammoniumalkylcarboxy, P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10)guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy.
 4. The compoundof claim 3, wherein the 3 of R₁ through R₁₄ independently selected fromthe group consisting of a substituted or unsubstituted (C1-C10)alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl,(C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkyl, a substituted or unsubstituted arylamino-(C1-C10) alkyl, asubstituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, and(C1-C10) quaternaryammoniumalkylcarboxy.
 5. The compound of claim 1,wherein the second steroid is a compound of formula I.
 6. The compoundof claim 1, wherein the linking group is (C1-C10) alkyl-oxy-(C₁-C10)alkyl.
 7. The compound of claim 1, wherein none of R₅, R₈, R₉, R₁₃, andR₁₄ is deleted.
 8. The compound of claim 1, wherein each of R₃, R₇, andR₁₂ is independently selected from the group consisting of a substitutedor unsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted(C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, a substituted or unsubstituted (C1-C10)aminoalkylcarboxamido, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkylcarboxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyloxy, and (C1-C10)guanidinoalkylcarboxy, where Q5 is a side chain of any amino acid, P.G.is an amino protecting group or a pharmaceutically acceptable saltthereof.
 9. The compound of claim 8, wherein R₁, R₂, R₄, R₅, R₆, R₈,R₁₀, R₁₁, R₁₃, R₁₄, R₁₅, and R₁₆ are hydrogen.
 10. The compound of claim9, wherein R₁₇ is —CR₁₈R₁₉R₂₀, where each of R₁₈, R₁₉, and R₂₀, isindependently selected from the group consisting of hydrogen, hydroxyl,a substituted or unsubstituted (C1-C10) alkyl, (C1-C10) hydroxyalkyl,(C1-C10) alkyloxy-(C1-C10) alkyl, a substituted or unsubstituted(C1-C10) aminoalkyl, a substituted or unsubstituted aryl, (C1-C10)haloalkyl, (C2-C6) alkenyl, (C2-C6) alkynyl, oxo, and a linking groupattached to a second steroid.
 11. The compound of claim 8, wherein eachof R₃, R₇, and R₁₂, is independently selected from the group consistingof —O—(CH2)n-NH2, —O—CO—(CH2)n-NH2, —O—(CH2)n-NH—C(NH)—NH2,—O—(CH2)n-N3, —O—(CH2)n-CN, where n is 1 to 3, and —O—C(O)—HC(Q5)-NH2,where Q5 is a side chain of any amino acid.
 12. The compound of claim 8,wherein each of R₃, R₇, and R₁₂, is —O—CO—(CH2)n-NH2, where n is 1 to 4.13. The compound of claim 12, wherein R17 is—CH(CH₃)(CH₂)₃—O—(CH₂)_(n)—NH2, wherein n is 1-7.
 14. The compound ofclaim 12, wherein R17 is —CH(CH₃)—(CH₂)_(n)—NR¹R², wherein n is 0-2, R¹and R² are independently (C1-C6) alkyl, aryl or aralkyl.
 15. Thecompound of claim 1, wherein R17 is —CH(CH₃)(CH₂)_(n1)—CO—OR³, where R³is selected from —CH₂)_(n2)N⁺(CH₃)₃, wherein n1 and n2 are independently1-4.
 16. The compound of claim 15, wherein R3, R7, and R12 are—O—C(O)—(CH₂)_(n)—NH₂, wherein n is 1-5.
 17. The compound of claim 1having the following formula:

wherein n is 1-3, and Bn is a benzyl group.
 18. The compound of claim 1having the following formula:

wherein n is 1-3, and R is selected from n-octyl, andtrimethylethylammonio.
 19. The compound of claim 1 having the formula:


20. A method of preparing the compound according to formula I

wherein fused rings A, B, C, and D are independently saturated or fullyor partially unsaturated; and R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆,and R₁₇ is each independently selected from the group consisting ofhydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,(C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10)alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl,(C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, asubstituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, asubstituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substitutedor unsubstituted (C1-C10) aminoalkylaminocarbonyl, a substituted orunsubstituted (C1-C10) aminoalkylcarboxamido, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyl oxy, (C1-C10)quaternaryammoniumalkylcarboxy, and (C1-C10) guanidinoalkyl carboxy,where Q5 is a side chain of any amino acid, P.G. is an amino protectinggroup, and R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is each independently: deletedwhen one of fused rings A, B, C, or D is unsaturated so as to completethe valency of the carbon atom at that site, or selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, alinking group attached to a second steroid, a substituted orunsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted(C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)—C(O)—O—,(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5is a side chain of any amino acid, P.G. is an amino protecting group,and provided that at least two of R₁ through R₁₄ are independentlyselected from the group consisting of a substituted or unsubstituted(C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl,(C1-C10) quaternaryammonium alkylcarboxy, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)—C(O)—O—, (C1-C10) guanidinoalkyloxy, and (C1-C10)guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof;the method comprising contacting a compound of formula IV,

where at least two of R₁ through R are hydroxyl, and the remainingmoieties on the fused rings A, B, C, and D are defined for formula I,with an electrophile to produce an alkyl ether compound of formula IV,wherein at least two of R₁ through R₁₄ are (C1-C10)alkyloxy; convertingthe alkyl ether compounds into an amino precursor compound wherein atleast two of R₁ through R₁₄ are independently selected from the groupconsisting of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy; andreducing the amino precursor compound to form a compound of formula I.21. The method of claim 20, wherein the electrophile is allylbromide.22. A method of producing a compound of formula I:

wherein fused rings A, B, C, and D are independently saturated or fullyor partially unsaturated; and R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, R₁₆,and R₁₇ is each independently selected from the group consisting ofhydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,(C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10)alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl,(C1-C10) alkylamino-(C₁-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted arylamino-(C1-C10) alkyl, (C1-C10) haloalkyl, C2-C6alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, asubstituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, asubstituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substitutedor unsubstituted (C1-C10) aminoalkylaminocarbonyl, a substituted orunsubstituted (C1-C10) aminoalkylcarboxamido, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyl oxy, (C1-C10)quaternaryammoniumalkylcarboxy, and (C1-C10) guanidinoalkyl carboxy,where Q5 is a side chain of any amino acid, P.G. is an amino protectinggroup, and R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is each independently: deletedwhen one of fused rings A, B, C, or D is unsaturated so as to completethe valency of the carbon atom at that site, or selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, alinking group attached to a second steroid, a substituted orunsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted(C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5is a side chain of any amino acid, P.G. is an amino protecting group,and provided that at least two of R₁ through R₁₄ are independentlyselected from the group consisting of a substituted or unsubstituted(C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10) alkyl, (C1-C10)alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkylcarboxy, a substituted or unsubstituted arylamino-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10)alkyl, a substituted or unsubstituted (C1-C10) aminoalkylaminocarbonyl,(C1-C10) quaternaryammonium alkylcarboxy, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyloxy, and (C1-C10)guanidinoalkylcarboxy; or a pharmaceutically acceptable salt thereof;the method comprising contacting a compound of formula IV,

where at least two of R₁ through R₁₄ are hydroxyl, and the remainingmoieties on the fused rings A, B, C, and D are defined for formula I,with an electrophile to produce an alkyl ether compound of formula IV,wherein at least two of R₁ through R₁₄ are (C1-C10) alkyloxy; convertingthe alkyl ether compound into an amino precursor compound wherein atleast two of R₁ through R₁₄ are independently selected from the groupconsisting of (C1-C10) azidoalkyloxy and (C1-C10) cyanoalkyloxy;reducing the amino precursor compound to produce an aminoalkyl ethercompound wherein at least two of R₁ through R₁₄ are (C1-C10)aminoalkyloxy; and contacting the aminoalkyl ether compound with aguanidino producing electrophile to form a compound of formula I. 23.The method of claim 22, wherein the guanidino producing electrophile isHSO₃—C(NH)—NH₂.
 24. A pharmaceutical composition comprising an effectiveamount of a compound of claim
 1. 25. The pharmaceutical composition ofclaim 24, wherein the composition includes additional antibiotics.
 26. Amethod of treating a microbial infection of a host by administering tothe host an effective amount of an anti-microbial composition comprisinga compound according to claim
 1. 27. The method of claim 26 wherein thehost is a human.
 28. The method of claim 26 wherein the anti-microbialcomposition further comprises a second anti-microbial substance to bedelivered into a microbial cell.
 29. The method of claim 28 wherein thesecond anti-microbial substance is an antibiotic.
 30. The method ofclaim 26 wherein the infection is a bacterial infection.
 31. The methodof claim 30 wherein the infection is a infection a Gram-negativebacterial infection.
 32. The method of claim 30 wherein the bacterialinfection is an infection with a bacterium characterized by an outermembrane comprising a substantial percentage of lipid A.
 33. A method ofenhancing cell permeability by administering to the cell apermeability-enhancing amount of the compound of claim
 1. 34. The methodof claim 33 further comprising administering to the cell a substance tobe introduced into the cell.
 35. The method of claim 34 in which thecell is a bacterium.
 36. The method of claim 35 in which the bacteriumis a Gram-negative bacterium.
 37. The method of claim 34 in which thecell is a sperm cell and the compound is part of a spermicidalcomposition.
 38. A method of identifying compounds effective against amicrobe comprising administering a candidate compound and a compoundaccording to claim 1 to the microbe and determining whether thecandidate compound has a static or toxic effect on the microbe.
 39. Themethod of claim 38 in which the microbe is a Gram-negative bacterium.40. A method of microbial growth control comprising contacting a microbewith an effective amount of anti-microbial composition comprising acompound according to claim
 1. 41. A composition of matter comprisingthe compound of claim 1 in combination with an anti-microbial substanceto be introduced into a cell.
 42. A compound comprising a ring system ofat least 4 fused rings, each of the rings having from 5-7 atoms, thering system having two faces, wherein the compound comprises 3 chainsattached to the same face of the ring system, each of the chainscontaining a multiple nitrogen-containing group, wherein the multiplenitrogen-containing group is separated from the ring system by at leastone atom, and wherein the multiple nitrogen-containing group is a(C1-C10) alkylamino (C1-C10) alkyamino group or a (C1-C10) alkylamino(C1-C10) alkyamino (C1-C10) alkyamino group.
 43. The compound of claim42, wherein each of the mulitiple nitrogen-containing groups isseparated from the steroid backbone by at least two atoms.
 44. Thecompound of claim 43, wherein each of the multiple nitrogen-containinggroups is separated from the steroid backbone by at least three atoms.45. The compound of claim 44, wherein each of the multiplenitrogen-containing groups is separated from the steroid backbone by atleast four atoms.
 46. The compound of claim 42, wherein the compoundfurther comprises a hydrophobic group attached to the steroid backbone.47. The compound of claim 42, wherein the hydrophobic group is selectedfrom the group consisting of a substituted (C3-10) aminoalkyl group, a(C1-10) alkyloxy (C3-10) alkyl group, and a (C1-10) alkylamino(C3-10)alkyl group.
 48. A pharmaceutical composition comprising aneffective amount of a compound of claim
 42. 49. A method of enhancingcell permeability by administering to the cell a permeability enhancingamount of the compound of claim
 42. 50. A compound of claim 1 having theformula:

wherein R₁ is selected from hydrogen, or (C1-C10) alkylamino, R₂ isselected from (C1-C10) alkylamino or (C1-C10) alkylamino-(C1-C10)alkylamino, and n is 1-3.
 51. The compound of claim 1, wherein R₁ ishydrogen and R₂ is (C1-C10) alkylamino-(C1-C10) alkylamino.
 52. Thecompound of claim 1, wherein R₁ is (C1-C10) alkylamino, and R₂ is(C1-C10) alkylamino.
 53. A compound according to formula I

wherein: fused rings A, B, C, and D are independently saturated or fullyor partially unsaturated; and R₁ through R₄, R₆, R₇, R₁₁, R₁₂, R₁₅, andR₁₆, is each independently selected from the group consisting ofhydrogen, hydroxyl, a substituted or unsubstituted (C1-C10) alkyl,(C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl, (C1-C10)alkylcarboxy-(C1-C10) alkyl, (C1-C10) alkylamino-(C1-C10) alkyl,(C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10) alkylamino-(C1-C10)alkylamino-(C1-C10) alkylamino, a substituted or unsubstituted (C1-C10)aminoalkyl, a substituted or unsubstituted aryl, a substituted orunsubstituted arylamino-(C₁-C10) alkyl, (C1-C10) haloalkyl, C2-C6alkenyl, C2-C6 alkynyl, oxo, a linking group attached to a secondsteroid, a substituted or unsubstituted (C1-C10) aminoalkyloxy, asubstituted or unsubstituted (C1-C10) aminoalkyloxy-(C1-C10) alkyl, asubstituted or unsubstituted (C1-C10) aminoalkylcarboxy, a substitutedor unsubstituted (C1-C10) aminoalkylaminocarbonyl, a substituted orunsubstituted (C1-C10) aminoalkylcarboxamido, H2N—HC(Q5)-C(O)—O—,H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy,P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10) guanidinoalkyl oxy, (C1-C10)quaternaryammoniumalkylcarboxy, and (C1-C10) guanidinoalkyl carboxy,where Q5 is a side chain of any amino acid, P.G. is an amino protectinggroup, and R₅, R₈, R₉, R₁₀, R₁₃, and R₁₄ is each independently: deletedwhen one of fused rings A, B, C, or D is unsaturated so as to completethe valency of the carbon atom at that site, or selected from the groupconsisting of hydrogen, hydroxyl, a substituted or unsubstituted(C1-C10) alkyl, (C1-C10) hydroxyalkyl, (C1-C10) alkyloxy-(C1-C10) alkyl,a substituted or unsubstituted (C1-C10) aminoalkyl, a substituted orunsubstituted aryl, C1-C10 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, alinking group attached to a second steroid, a substituted orunsubstituted (C1-C10) aminoalkyloxy, a substituted or unsubstituted(C1-C10) aminoalkylcarboxy, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—,(C1-C10) azidoalkyloxy, (C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—,(C1-C10) guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy, where Q5is a side chain of any amino acid, P.G. is an amino protecting group,and R17 is selected from the group consisting of substituted orunsubsituted alkylcarboxyalkyl and protected or unprotectedpoly(aminoalkyl), provided that at least two of R₁ through R₁₄ areindependently selected from the group consisting of a substituted orunsubstituted (C1-C10) aminoalkyloxy, (C1-C10) alkylcarboxy-(C1-C10)alkyl, (C1-C10) alkylamino-(C1-C10) alkylamino, (C1-C10)alkylamino-(C1-C10) alkylamino-(C1-C10) alkylamino, a substituted orunsubstituted (C1-C10) aminoalkylcarboxy, a substituted or unsubstitutedarylamino-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkyloxy-(C1-C10) alkyl, a substituted or unsubstituted (C1-C10)aminoalkylaminocarbonyl, (C1-C10) quaternaryammonium alkylcarboxy,H2N—HC(Q5)-C(O)—O—, H2N—HC(Q5)-C(O)—N(H)—, (C1-C10) azidoalkyloxy,(C1-C10) cyanoalkyloxy, P.G.-HN—HC(Q5)-C(O)—O—, (C1-C10)guanidinoalkyloxy, and (C1-C10) guanidinoalkylcarboxy; or apharmaceutically acceptable salt thereof.
 54. The compound of claim 53,wherein the compound has the formula:

wherein n is 1-3.
 55. The compound of claim 53, wherein the compound hasthe formula:

wherein n is 1-3.
 56. Canceled.
 57. Canceled.
 58. Canceled.